Hypophosphatemic osteosclerosis, hyperostosis, and enthesopathy associated with novel homozygous mutations of DMP1 encoding dentin matrix protein 1 and SPP1 encoding osteopontin: The first digenic SIBLING protein osteopathy?

Journal Pre-proof Hypophosphatemic osteosclerosis, hyperostosis, and enthesopathy associated with novel homozygous mutations of DMP1 encoding dentin matrix protein 1 and SPP1 encoding osteopontin: The first digenic SIBLING protein osteopathy?

Michael P. Whyte, S. Deepak Amalnath, William H. McAlister, Marc D. McKee, Deborah J. Veis, Margaret Huskey, Shenghui Duan, Vinieth N. Bijanki, Suhas Alur, Steven Mumm PII:

S8756-3282(19)30486-7

DOI:

https://doi.org/10.1016/j.bone.2019.115190

Reference:

BON 115190

To appear in:

Bone

Received date:

16 October 2019

Revised date:

10 December 2019

Accepted date:

12 December 2019

Please cite this article as: M.P. Whyte, S.D. Amalnath, W.H. McAlister, et al., Hypophosphatemic osteosclerosis, hyperostosis, and enthesopathy associated with novel homozygous mutations of DMP1 encoding dentin matrix protein 1 and SPP1 encoding osteopontin: The first digenic SIBLING protein osteopathy?, Bone(2019), https://doi.org/ 10.1016/j.bone.2019.115190

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Hypophosphatemic Osteosclerosis, Hyperostosis, And Enthesopathy Associated With Novel Homozygous Mutations Of DMP1 Encoding Dentin Matrix Protein 1 and SPP1 Encoding Osteopontin: The First Digenic SIBLING Protein Osteopathy? Michael P. Whyte,1,2 S. Deepak Amalnath,3 William H. McAlister,4 Marc D. McKee,5 Deborah J. Veis,1,2 Margaret Huskey,2 Shenghui Duan,2 Vinieth N. Bijanki,1 Suhas Alur,3 Steven Mumm1,2 1

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Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children - St. Louis; St. Louis, MO 63110, USA

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Division of Bone and Mineral Diseases, Department of Internal Medicine, Washington University School of Medicine at Barnes-Jewish Hospital; St. Louis, MO 63110, USA

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Department of Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research; Pondicherry, 605006, India

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Mallinckrodt Institute of Radiology, Washington University School of Medicine at St. Louis Children’s Hospital; St. Louis, MO 63110, USA

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Faculty of Dentistry and Department of Anatomy and Cell Biology, McGill University; Montreal, Quebec, H3A 0C7, Canada

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Funded in part by: Shriners Hospitals for Children, the Clark and Mildred Cox Inherited Metabolic Bone Disease Research Fund and the Hypophosphatasia Research Fund at the BarnesJewish Hospital Foundation, the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (NIH) under Award DK067145, and the Canadian Institutes for Health Research under Award MOP-142330. The content of the manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Presented in part at: American Society for Bone and Mineral Research 2019 Annual Meeting, September 20-23, 2019; Orlando, FL, USA [J Bone Miner Res 34 (Suppl. 1): 121-122, 2019]. Key Words: ABCC6, autosomal recessive hypophosphatemic rickets, CYP27B1, endogamy, FGF23, heterotopic ossification, hydroxyapatite, hypophosphatemia, inorganic phosphate, iron deficiency, mineralization, osteomalacia, phosphatonin, rickets, scoliosis, tooth loss Running Title: Digenic SIBLING Osteopathy

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Journal Pre-proof Correspondence To: Michael P. Whyte, M.D. Shriners Hospitals for Children-St. Louis 4400 Clayton Avenue St. Louis, MO 63110, USA Telephone: 314-872-8305 Disclosures: The authors have no conflict of interest.

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E-Mail Addresses: Michael P. Whyte – [email protected] S. Deepak Amalnath –[email protected] William H. McAlister – [email protected] Marc D. McKee –[email protected] Deborah J. Veis - [email protected] Margaret Huskey – [email protected] Shenghui Duan – [email protected] Vinieth N. Bijanki – [email protected] Suhas Alur – [email protected] Steven Mumm – [email protected]

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Abstract: 269 words Text: 5372 words Figures: 8 Tables: 1

Highlights:     

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Author Contributions: All authors helped write and approved the submitted manuscript. MPW guided some patient studies and drafted and finalized the report. SDA with SA identified the patients, characterized their disorder, and made possible the mutation analyses. WHM delineated the radiological findings. MDM performed the bone immunohistochemistry and interpreted the osteopontin excess. DJV evaluated the decalcified bone histopathology. VNB helped reference and illustrate the manuscript. MH and SD conducted the mutation analyses interpreted by SM.

SIBLINGs (DMP1, SPP1 or OPN, IBSP or BPS, MEPE, and DSPP) condition mineralization To date, two monogenic disorders represent the SIBLING genes DMP1 and DSPP DMP1 deactivation causes autosomal recessive hypophosphatemic rickets, 1 (ARHR1) An endogamous Indian family with ARHR1 harbors homozygous DMP1 and SPP1 mutations Possibly, they represent the first example of a digenic SIBLING protein osteopathy

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I)

Abstract: The SIBLINGs are a subfamily of the secreted calcium-binding phosphoproteins and

comprise five small integrin-binding ligand N-linked glycoproteins [dentin matrix protein-1 (DMP1), secreted phosphoprotein-1 (SPP1) also called osteopontin (OPN), integrin-binding sialoprotein

(IBSP)

also

called

bone

sialoprotein

(BPS),

matrix

extracellular

phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (DSPP)]. Each SIBLING has at

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least one “acidic, serine- and aspartic acid-rich motif” (ASARM) and multiple Ser-x-Glu/pSer

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sequences that when phosphorylated promote binding of the protein to hydroxyapatite for

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regulation of biomineralization. Mendelian disorders from loss-of-function mutation(s) of the

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genes that encode the SIBLINGs thus far involve DSPP causing various autosomal dominant

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dysplasias of dentin but without skeletal disease, and DMP1 causing autosomal recessive hypophosphatemic rickets, type 1 (ARHR1). No diseases have been reported from gain-of-

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function mutation(s) of DSPP or DMP1 or from alterations of SPP1, IBSP, or MEPE. Herein, we describe severe hypophosphatemic osteosclerosis and hyperostosis associated

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with skeletal deformity, short stature, enthesopathy, tooth loss, and high circulating FGF23 levels

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in a middle-aged man and young woman from an endogamous family living in southern India. Both shared novel homozygous mutations within two genes that encode a SIBLING protein: stop-gain (“non-sense”) DMP1 (c.556G>T, p.Glu186Ter) and missense SPP1 (c.769C>T, p.Leu266Phe). The man alone also carried novel heterozygous missense variants within two additional genes that condition mineral homeostasis and are the basis for autosomal recessive disorders: CYP27B1 underlying vitamin D dependent rickets, type 1, and ABCC6 underlying both generalized arterial calcification of infancy, type 2 and pseudoxanthoma elasticum (PXE). By immunochemistry, his bone contained high amounts of OPN, particularly striking

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Journal Pre-proof surrounding osteocytes. We review how our patients’ disorder may represent the first digenic SIBLING protein osteopathy.

II)

Introduction: The SIBLING proteins comprise the five small integrin-binding ligand N-linked

glycoproteins [dentin matrix protein-1 (DMP1), secreted phosphoprotein-1 (SPP1) also called osteopontin (OPN), intergrin-binding sialoprotein (IBSP) also called bone sialoprotein (BSP),

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matrix extracellular phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (DSPP)]

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while representing a subfamily of the secreted calcium-binding phosphoproteins.(1)

Each

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SIBLING protein has at least one “acidic, serine- and aspartic acid-rich motif” (ASARM) as well

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as multiple Ser-x-Glu/pSer sequences.(1) When phosphorylated, these bind free calcium (Ca) or the Ca in hydroxyapatite (HA) crystals and, modulated by their phosphorylation status,

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importantly regulate skeletal and dental mineralization.(2)

disease.(3)

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Currently among the five SIBLING proteins, two are causally related to heritable Autosomal recessive (AR) hypophosphatemic rickets, type 1 (ARHR1: OMIM

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#241520)(3) involves loss-of-function mutations of DMP1(4-12) whereas autosomal dominant

mutations

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(AD) forms of dentinogenesis imperfecta without skeletal disease involve loss-of-function

of DSPP.(13-16) Mendelian diseases have not been attributed to gain-of-function mutation(s) of DSPP or DMP1 or to mutations that alter SPP1, IBSP, or MEPE.(3) ARHR1 features diffuse osteosclerosis including a coarse and thickened calvarium and broad and poorly-mineralized ribs and clavicles together with enthesopathy, but few individuals with ARHR1 verified by DMP1 sequencing are reported in the medical literature,(4-12) and few publications detail the disorder’s clinical, biochemical, radiological, or histopathological findings.(7,8)

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Journal Pre-proof Herein, we delineate severe generalized osteosclerosis and hyperostosis (i.e., trabecular and cortical bone thickening, respectively),(17) tooth loss, and marked enthesopathy with FGF23mediated hypophosphatemia in a middle-aged man and a young woman from an endogamous family living in southern India. Both had severe skeletal deformities and short stature consistent with rickets during their childhood and were found to share novel homozygous mutations within DMP1 and SPP1. He also carried novel heterozygous missense variants within two additional

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genes associated with mineral homeostasis; CYP27B1 underlying vitamin D-dependent rickets,

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type 1 (VDDR1) and ABCC6 underlying the allelic disorders generalized arterial calcification of

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infancy (GACI), type 2 and pseudoxanthoma elasticum (PXE).(3) Our two patients represent the

Patients and Methods:

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III)

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first with mutated OPN, and possibly the first digenic SIBLING protein osteopathy.

A. Family:

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The two affected individuals were from an endogamous family living in the state of

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Tamil Nadu in southern India, with the propositus’ father having had marriages that were

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consanguineous to two women (Figure 1).

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Journal Pre-proof Figure 1:

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Family Pedigree:

Neither individual had been evaluated for skeletal disease, took medications, or used supplements. Both began losing teeth as teenagers, ate the regional diet, and had short stature whereas their siblings reportedly had normal heights.

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Journal Pre-proof B. Case Reports: 1) Patient 1: The propositus (III-2, Figure 1), 44 years-of-age, reported himself well until age 10 years when frontal bossing and bowing of his legs appeared and progressed. By age 20 years he was considered “physically challenged” and received special vocational training and then bicycled to work at a small factory where he cut steel sheets and rods without toxin exposure. His right hand was damaged in a work accident. At age 40 years he was struck

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by an automobile but did not sustain major injuries including fractures. However, walking

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became progressively difficult and he presented to the Department of Medicine, Jipmer, India in Computed

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2014 with spastic quadriparesis involving especially his lower extremities.

tomography (CT) and magnetic resonance (MR) imaging showed compressive myelopathy of his

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cervical spinal cord due to widened sclerotic vertebrae C4-C6 (see Radiological Findings).

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Despite cervical laminectomy with biopsy of a vertebra (see Histopathological Findings), his

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symptoms worsened and spasticity and severe back pain limited walking to just several steps. Physical examination upon referral showed considerable kyphosis and frontal bossing of

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his skull (Figure 2). Hearing was clinically normal. He was edentulous and shorter, standing

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145 cm (4 ft., 9 in.), than his mother who appeared of normal height.

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Figure 2:

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Patient 1:

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At age 44 years, the propositus’ skull a,b) is enlarged with b) marked frontal bossing. He required a walker c) for severely limited ambulation. 2) Patient 2: This woman (III-5, Figure 1), age 25 years, reported that her skeletal

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deformities began at about 8 years-of-age. She was recognized to have the propositus’ disorder

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when our mutation analyses revealed she carried his homozygous DMP1 and SPP1 mutations (see Mutation Analyses). She was ambulant, of normal intelligence, cared for herself without

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assistance, and reported no deafness.

Physical examination showed less cranial and extremity deformity than manifested by the propositus, but especially severe kyphoscoliosis was present since childhood and markedly compromised her height measuring 120 cm (3 ft, 11 in) (Figure 3).

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Journal Pre-proof Figure 3:

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Patient 2:

At age 25 years, Patient 2’s skull a,b) shows mild frontal bossing. There is c) severe kyphoscoliosis, and d,e) deformed lower limbs with long bone bowing.

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Journal Pre-proof C. Biochemical Findings: Routine biochemical studies, including assessments of mineral and skeletal homeostasis, were performed by the Department of Medicine in Pondicherry, India. Serum C-terminal FGF23 was quantitated using an ELISA at P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India. D. Radiological Findings:

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All radiological studies, including the propositus’ CT and MR imaging, were reviewed

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and the findings contrasted between both patients and then with images of ARHR1 in the

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medical literature. E. Histopathological Findings:

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The bone specimen obtained during Patient 1’s cervical laminectomy was fixed in 10%

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formalin and 5% nitric acid, decalcified as assessed tangibly, embedded in paraffin, and then

(see below).

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stained with hematoxylin and eosin. Additional sections were cut for OPN immunochemistry

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F. Mutation Analyses:

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Informed written consent from both patients, including as indicated by the Human Research Protection Office, Washington University School of Medicine, St. Louis, MO, USA, preceded peripheral blood sampling to extract leukocyte DNA for candidate gene mutation analysis. The DNA was isolated using the phenol/chloroform method and forwarded using express mail to our research laboratory in St. Louis, MO, USA. Because early on the propositus’ marked hyperphosphatasemia and severe skeletal disease (see Results) seemed reminiscent of juvenile Paget’s disease,(18-20) we initially Sangersequenced: i) all the coding exons and adjacent mRNA splice sites of TNFRSF11B encoding

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Journal Pre-proof osteoprotegerin (OPG),(19) and ii) exon 1 of TNFRSF11A encoding receptor activator of NF-κB (RANK).(20) After negative findings, Ion Torrent (Thermo Fisher Scientific, Waltham, MA, USA) next generation sequencing (NGS) assessed 35 genes: i) involved in elevated bone mass disorders, ii) that condition human skeletal remodeling, and iii) which underlie mouse models resembling his skeletal phenotype: TNFRSF11A (RANK) and TNFRSF11B (OPG) as well as TNFSF11 (RANKL), VCP, SQSTM1, TGFB1, IFITM5, MAFB, CSF1, CSFR1, TRAF6, RELA,

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RELB, REL, NFKB1, NFKB2, TFEB, CA2, CLCN7, CTSK (CATHEPSIN K), OSTM1,

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PLEKHM1, TCIRG1, SOST, SLC29A3, LRP4, LRP5, LRP6, SNX10, FAM20C, FAM123B

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(AMER1), TYROBP, LEMD3, DLX3, and PTDSS1. When these findings too were negative and

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marked hypophosphatemia, renal inorganic phosphate (Pi) wasting, and elevated serum FGF23 were recognized, we performed Ion Torrent NGS using our panel of 15 genes involved in Pi and

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inorganic pyrophosphate (PPi) metabolism and/or that cause or are candidate genes for other

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heritable forms of rickets: ABCC6, ALPL (TNSALP), ANKH, CYP27B1, DMP1, ENPP1, FGF23, GALNT3, KL (KLOTHO), MEPE, PHEX, PHOSPHO1, SLC34A3, SPP1 (OPN), and VDR. The

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four missense variants identified (see Results) in his DMP1, SPP1, CYP27B1, and ABCC6 were

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verified by Sanger-sequencing and then searched for in Patient 2. Both Ion Torrent panels assessed all coding exons and at least 10 bp of the flanking introns to encompass sequences important for mRNA splicing. For a few genes (TNFRSF11A, TNFRSF11B, TNFSF11, IFITM5, ALPL, ANKH, DMP1, ENPP1, FGF23, and PHEX), we also examined both the 5'- and 3'-UTR. Our PCR conditions and primer sequences are available upon request. The genetic variants (see Results) in the two patients were evaluated for their frequency in

large

populations

using

the

Exome Aggregation

Consortium

(ExAC)

Browser

(exac.broadinstitute.org) and in ClinVar (www.ncbi.nlm.nih.gov/clinvar/). The impact of the

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Journal Pre-proof missense variants on protein function was predicted using the Sorting Intolerant From Tolerant (SIFT) web server(21) and Polymorphism Phenotyping v2 (PolyPhen-2).(22) G. Osteopontin Immunohistochemistry: Paraffin sections from the propositus’ cervical vertebra specimen were dewaxed in xylene, hydrated in an ethanol series to pure distilled water, and then quenched of endogenous peroxidase activity with 3% hydrogen peroxide (Fisher Scientific, Hampton, NH, USA).

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Blocking was performed using 1% horse normal serum (Vector Laboratories, Burlingame, CA,

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USA) for 30 min at 37°C. Sections were then incubated for 1 hr at 37°C with goat anti-human

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OPN antibody (R&D Systems, Minneapolis, MN, USA) diluted 1:20 in 1X PBS-0.1% Tween.

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After washing, the sections were incubated for 30 min at 37°C with secondary biotinylated horse anti-goat IgG (Vector Laboratories) diluted 1:200 in 1X PBS. Biotinylated secondary antibody

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was then captured by Avidin/Biotinylated enzyme Complex (ABC complex) (Vector

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Laboratories). Finally, antibody staining was visualized by a peroxidase activity detection kit using AEC Peroxidase Substrate (Vector Laboratories). Control incubations to evaluate

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nonspecific staining followed the same procedure except that the primary antibody was omitted Additional control

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and instead substituted with 1X PBS-0.1% Tween antibody diluent.

incubations were performed on sound, age-matched bone samples collected from control patients having non-XLH-related surgical procedures.

IV)

Results: A. Biochemical Findings: Routine studies of mineral metabolism showed both patients were hypophosphatemic

with reduced TmP/GFR attributable to high circulating levels of FGF23 (Table). Their serum alkaline phosphatase (ALP) activity was elevated, and highest in Patient 1. Patient 2, whose

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Journal Pre-proof blood hemoglobin level was low at 10 g/dl (Nl, 11-15), had an especially high FGF23 concentration, perhaps partly attributable to iron deficiency.(23) concentration was deficient.

Also, her serum 25(OH)D

Both individuals were mildly hypocalcemic, which is not

uncommon in Pondicherry, India.

Parathyroid hormone (PTH) levels were not measured.

Relatively low Pi levels in both patients’ urine despite renal Pi wasting likely reflected little dietary Pi.

Thus, several environmental factors (little sunshine exposure and poor dietary

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mineral content) perhaps impacted their mineral metabolism and skeletal phenotype (see

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Discussion).

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Table 1 Clinical Laboratory Findings Test

Units

Patient 1

Patient 2

Reference Range*

Serum mg/dl ″

8.3 1.6

8.9 1.7

9 – 11 2.5 - 4.5

25(OH) vitamin D Alkaline phosphatase FGF23 Urea Creatinine

ng/ml IU/L RU/ml mg/dl ″ ″

ND‡ 1,249 276 15 0.8 3.2

7.3 350 1,978 18 0.7

Magnesium Sodium

″ meq/L

1.9 138

20 – 100 30 – 125 0 – 150 15 – 40 0.7 - 1.2 2.5 - 7.0 ♂ 1.5 - 6.0 ♀ 1.8 - 3.0 135 – 145

Potassium Chloride AST ALT GGT Total Protein

″ ″ IU/L ″ ″ mg/dl

4.0 105 28 21 22 6.8

Albumin Bilirubin total (direct) Total cholesterol Triglycerides HDL LDL

g/dl mg/dl ″ ″ ″ ″

Hemogram Hemoglobin White Blood Cells Platelets Prothrobin Time

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ND 0.4 ND ″ ″ ″

4.3 0.5 (0.2) 151 81 40 95

3.5 - 5.5 0.4 - 1.2 < 200 < 150 > 40 < 100

″

″

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< 30

g/dl x103/ul Kx103/ul

13.2 7,700 102 ND

10.5 5,600 120 14.9

11 – 15 3.8 – 9.8 140 – 440 9.1 – 12.0

ND

1.10

0.8 – 1.2

%

90

46

78-91

mmol/L

0.51

0.25

0.90 - 1.35

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3.5 – 5.0 96 – 106 0 – 40 0 – 45 1 – 50 6.3 - 8.3

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VLDL

1.0 ND 138 3.8 ND 25 19 13 6.7

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Uric acid

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Calcium Phosphorus (Pi)

INR Urine Tubular reabsorption PO4 (TRP) TmP/GFR * Adults ‡ ND = Not done.

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Journal Pre-proof B. Radiological Findings: 1) Patient 1: We evaluated the radiographic views of his lateral skull and cervical spine; anteroposterior (AP) and lateral thoracolumbar spine; AP chest, pelvis, femurs, and legs; and single views of both upper extremities as well as the MR imaging of his head and spine, and CT of his neck. The radiographic abnormalities were numerous (Figure 4 and Supplementary

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Appendix). The skull was diffusely osteosclerotic with a thickened diploic space, prominent

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frontal region, maxillary hypoplasia, expanded ascending ramus of the mandible, and large

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external occipital protuberance. His entire spine was osteosclerotic, including the vertebral

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bodies and neural arches, yet with mild vertebral body compressions and denser margins. There were extensive areas of enthesopathy, osteophytosis, and ligamentous ossification with spinal

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canal narrowing, especially in the cervical region. Obliteration of some of the apophyseal joints

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and ankylosis across the intervertebral discs were apparent. The ribs and clavicles were expanded with thick cortices and trabeculae.

His pelvis showed flared iliac wings, deep

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acetabular sockets, protrusio acetabuli, extensive osteophytosis and enthesopathy, and

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osteoarthritis of the hips with narrowed joint space. The tubular bones of the limbs were shortened, wide, deformed, and curved with cortical thickening and marked enthesopathy. His distal femora had trapezoidal ends characteristic of X-linked hypophosphatemia (XLH) in adults.(24) Interosseous fusions involved the radius and ulna as well as the tibia and fibula. Bony fusions also affected the hind feet and ankles. MR of his head and CT of his entire spine showed in the cervical spine very thick diploic space indenting the brain, which otherwise looked normal for age except for mild dilation of the lateral ventricles, a large uncommon sphenoid meningocele, and narrowing of the auditory

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Journal Pre-proof canals. His cervical vertebral bodies and neural arches were filled with osseous material without normal marrow. The lamina of the cervical spine were markedly expanded causing severe stenosis of canal with spinal cord compression.

The cervical spine also had osteophyte

formation, ligamentous ossification, and extensive enthesopathy. Severe stenosis compromised the auditory canals, but not the optic foramena. The occipital protuberance was large. His ribs,

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rather than osseous material. Kidney images were normal.

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clavicles, and mandible had a thick cortex and, unlike the skull and spine, were filled with fat

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Figure 4: Patient 1 Radiological Images:

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Legend to Figure 4: a) The lateral skull is extremely osteosclerotic with marked uniform diploic thickening, prominent frontal bone, hypoplastic maxilla, and enlarged external occipital protuberance. The ascending ramus of the edentulous mandible is expanded and prominent anteriorly and inferiorly. The cervical spine is osteosclerotic with marked osteophytosis and osseous bridging of the vertebrae with ligamentous ossification and a severely narrowed spinal canal. b) The ribs and clavicles are wide, sclerotic, and have coarse trabeculae. Extensive enthesopathy includes small bony excrescences from the ribs, clavicles, and the sclerotic scapulae. c) In the thoracic and lumbar spine, the vertebral bodies are sclerotic due to dense margins, but with osteopenic centers. d) The disc spaces are expanded with bowing into the vertebral bodies, and the spinal canal is narrow with expanded sclerotic neural arches. Extensive enthesopathy includes small bony proliferations from many bony surfaces, but also with extensive osteophytes and osseous bridging of the vertebral bodies and neural arches. Proliferations of bone are in the humerus at the deltoid insertions and on the medial humerus. e) The upper extremities show bone shortening, cortical thickening, widened medullary cavities with thick course trabeculae, and fusion of the interosseous membranes between the radius and ulna. There are post traumatic changes in the medial metacarpals and phalanges. f) The carpal bones are sclerotic, have degenerative changes, and extensive small bony proliferations of enthesopathy. Tubular bones of the hands are wide with degenerative changes greater on the right, and at the proximal interphalangeal joints the middle phalanges are wider than the proximal phalanges. Osseous fusion joins the 2nd metacarpal and the thumb and between the 5th metacarpal and phalanges. Scattered areas of enthesopathy, consisting of small bony proliferations, are present. g) The pelvis has flared iliac wings and extensive enthesopathy with bony proliferations from all pelvic bones including the ischia. Acetabular protrusio and degenerative changes in the hips are present with proliferations of bone at the acetabular margins and elsewhere. h) The femurs are short, wide, and trapezoidal at their distal ends, a characteristic of X-linked hypophosphatemia.(24) Wide femoral medullary cavities have coarse thick trabeculation. Enthesopathy with proliferations of bone is present along the external cortical surfaces. The knee joints are irregular with oblique joint surfaces. i,j,k) Old fracture deformities are present in the left distal tibia and fibula and the proximal right tibia, which are short with marked irregular cortical thickening, widening of the medullary cavities, and irregular proliferations of bone along their surfaces. There is bony fusion of the distal tibia and fibula, and fusion at the ankle joints. k) Fusions of the bones of the hindfoot include the talus, calcaneus, cuboid, and cuneiforms. Tarsometatarsal fusion is present with multiple small proliferations of bone off of many surfaces. l) MR shows the brain compressed by a thick diploic space. m) A single slice sagittal MR of the cervical spine and skull base shows a narrow spinal canal compressing the cervical cord and sclerotic expanded neural lamina. A portion of the sphenoid meningocele can be seen (asterisk). The vertebrae contain no fat as is apparent in the subcutaneous tissue. n,o) Axial CT images show sclerotic cervical vertebrae and a narrow spinal canal.

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Journal Pre-proof 2) Patient 2: This young woman was studied only radiographically. She had less severe skeletal disease in her skull and tubular bones than Patient 1, including less peripheral enthesopathy, however her kyphoscoliosis was much worse (Figure 5). Figure 5:

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Patient 2 Radiographic Images:

a) Lateral skull shows osteosclerosis, a thick diploic space, hypoplastic maxilla, and expanded mandible with abnormal teeth with prominent pulp chambers. The cervical spine has sclerotic vertebrae, a narrow spinal canal, and expanded lamina. b,c) Spine AP and lateral views show marked kyphoscoliosis suggestive of neuromuscular disease, some osteosclerosis, multiple osteophytes, and osseous and ligamentous fusions. d) Left lateral bending radiograph shows the osseous fusions in the lumbar spine with a “bamboo” appearance. Some straightening of the thoracic spine is apparent. The pelvis is tilted, has flared iliac wings, and extensive enthesopathy with small bony excrescences from many surfaces including the ischial bones. e) The femora show trapezoid appearance distally, widening, some cortical thickening, and many small bony excrescences of enthesopathy. f) The hands and forearms have sclerotic bone ends, course trabeculae, some cortical thickening, and extensive small areas of bony proliferations consistent with enthesopathy.

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Journal Pre-proof C. Bone Histopathology: Acid decalcification of patient 1’s cervical vertebra specimen precluded a histopathological diagnosis of osteomalacia. Furthermore, nuclear staining was not present to identify the various cell types. The specimen revealed fairly dense lamellar bone, with cortex and cancellum indistinguishable from each other (Figure 6). Figure 6:

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Patient 1 Decalcified Vertebral Histopathology:

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a) Low power image shows lack of demarcation of cortical vs. cancellous bone (periosteal surface at bottom right). Few marrow elements are present. b) Higher power image shows unremarkable bone with vascular channels. c) Polarization of the same field as in b) demonstrates well-organized lamellae. Decalcification precluded a histopathological diagnosis of osteomalacia. Scale bar (a) 500 μm, (b,c) 200 μm. Stain: hematoxylin and eosin.

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D) Osteopontin Immunochemistry: After decalcification, Patient 1’s cervical vertebra showed strong OPN staining by immunohistochemistry throughout the bone (Figure 7). Some regions that were more normallooking (albeit with sparse osteocytes) showed diffuse, strong immunostaining for OPN throughout the matrix, whereas other regions showed a very characteristic pattern of accumulation of OPN (reminiscent of that seen in XLH).(24) This striking distribution of OPN surrounding osteocytes, where the protein is particularly abundant in their peri-cellular (perilacunar) matrix and surrounding osteocyte dendritic cell processes by lining the canalicular

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Journal Pre-proof walls, is a hallmark distribution of XLH bone where diminished PHEX activity results in this accumulation pattern.(22,25) In these regions, some osteocytes and their surrounding matrix seemed normal in terms of OPN distribution. As OPN is a prominent constituent of cement lines observed in histological sections after immunolabeling,(26) cement lines were readily discernable related to both types of bone we observed, thus indicating at least some degree of bone

Figure 7:

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Patient 1: Bone Osteopontin Immunohistochemistry

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remodeling.

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a, b) Immunohistochemical staining (red) for osteopontin (OPN) in cervical vertebral bone. Bone remodeling is evident from the appearance of cement lines (CL) separating seemingly normal bone (white asterisks) having sparse osteocytes compared to overtly abnormal bone (black asterisks) showing abundant peri-osteocytic lesions ("halos" - similar to those seen in XLH,(25) brackets), with some nearby otherwise normal-looking osteocytes/lacunae (arrows) again as frequently seen in XLH. Intense OPN staining appears primarily associated with the bulk bone matrix of the more normal-looking areas, cement lines, and the peri-osteocytic lesions, and in the latter case the strong OPN staining extending as well into the fine canaliculi radiating from the abnormal osteocytes. c) Control incubation for OPN on non-XLH surgical waste bone, showing expected immunostaining predominantly in cement lines. d) Control incubations on a serial section where OPN primary antibody was omitted. Scale bars equal 50 µm.

Journal Pre-proof E. Mutation Analyses: Based on the propositus’ hyperphosphatasemia and skeletal findings, we first Sangersequenced all coding exons and adjacent mRNA splice sites of TNFRSF11B encoding OPG and exon 1 of TNFRSF11A encoding RANK, targeting a heritable rapid-remodeling skeletal disorder.(18) However, testing was negative. Then, no unequivocal disease-causing variants were found by Ion Torrent NGS analysis of 35 genes involved in high turnover or osteosclerotic

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diseases or mouse models with similar phenotypes. Instead, Ion Torrent NGS of 15 genes

i) a homozygous stop-gain (“nonsense”) change in DMP1 (c.556G>T,

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notable variants:

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involved in Pi and PPi metabolism and/or genetic forms of rickets revealed in both patients

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p.Glu186Ter) predicted to truncate the protein at amino acid residue 186 of the 513 total, and ii) a homozygous missense change in SPP1 encoding OPN (c.796C>T, p.Leu266Phe). The DMP1

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variant is pathogenic (PVS1, pathogenic very strong, null variant; PM2, pathogenic moderate,

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absent from controls; and PP1, pathogenic supporting, co-segregation with disease in multiple affected family members) according to American College of Medical Genetics (ACMG)

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Standards and Guidelines.(27) The SPP1 variant is reported at very low frequency (0.0009391) in

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ExAC (rs146563765) and is a non-conservative amino acid change implicating it to be a mutation. Nevertheless, it was predicted by SIFT(21) to be “tolerated” (score 0.11) and PolyPhen2(22) to be “benign” (score 0.016). Based on insufficient information at this time, it is considered a variant of uncertain significance by ACMG Standards and Guidelines. Exclusively in the propositus, Ion Torrent NGS also identified a heterozygous missense CYP27B1 variant (c.98C>T, p.Ala33Val) and a heterozygous ABCC6 variant (c.752G>A,p.Arg251Gln). His four gene variants were verified by Sanger sequencing (Figure 8). The heterozygous CYP27B1 variant was not reported as a mutation in ClinVar, or SNP in ExAC, making it unique and

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Journal Pre-proof predicting a conservative amino acid change assessed by SIFT to be “tolerated” (score 1.00)(21) and by PolyPhen-2 to be “benign” (score 0.002).(22) His ABCC6 variant was not found in ClinVar, and the predicted non-conservative amino acid change was not found in ExAC, suggesting potential impact on the protein’s function. However, it was predicted by SIFT to be “tolerated” (score 0.39),(21) and by PolyPhen-2 to be “benign” (score 0.004).(22) The CYP27B1 and ABCC6 variants did not clearly fit the ACMG guidelines(27) for benign variants and due to

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their rarity may be considered variants of unknown significance (VUS).

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Journal Pre-proof Figure 8:

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Mutations Detected In Patients 1 and 2 By Ion Torrent And Validated By Sanger Sequencing:

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a) Sanger sequencing DNA electropherograms of DMP1, SSP1, CYP27B1, and ABCC6 of both patients. The mutation/variant, when present, is listed above each electropherogram. Below each electropherogram, the DNA and amino acid sequences are shown with mutations in bold font. -------------------------------------------------Hom = homozygous. Het = heterozygous. b) Schematic of DMP1 gene (above) and mutant (below). Coding regions are shaded in blue. c) Amino acid sequence of DMP1 protein. Green line represents the location of termination (shaded amino acids are missing from the mutant protein). FAM20C phosphorylation sites (S-x-E motifs) appear in red.

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V)

Discussion: Both our patients manifested osteosclerosis and hyperostosis, enthesopathy, tooth loss,

and findings in keeping with severe rickets during childhood together with elevated circulating levels of FGF23, renal Pi wasting, and hypophosphatemia. We especially appreciated this important resemblance to ARHR1 when we discovered their novel homozygous stop-gain mutation within DMP1 predicted to render its encoded SIBLING protein non-functional. In fact,

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homozygous frameshift and splice site defects that compromise the coding region of DMP1

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cause ARHR1.(4-12) However, as discussed below, our patient’s dento-osseous disease seemed

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more severe than described in some of the few detailed reports of ARHR1.(4-12) Possibly, adverse environmental factors impacted their hard tissues, but multigenic AR disease is

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particularly prevalent in endogamous populations.(28) Early on, such genetic complexity was

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clarified using homozygosity mapping, positional cloning, etc. to identify the causal genes.

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Now, next-generation sequencing (NGS) more often provides the key information.(28) Our NGS panel of genes that underlie hypophosphatemic bone diseases and regulate Pi and PPi

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metabolism revealed that both our patients harbored not only a unique homozygous DMP1

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mutation, but also a unique homozygous variant within SPP1 predicted to alter their OPN. However, no mutation of SPP1 has been reported,(3) and therefore no additional phenotype could be anticipated other than hypermineralized bone sites revealed by OPN-deficient mouse studies.(29) Yet, OPN functions in processes as diverse as bone morphogenesis, skeletal and tooth mineralization, heart failure, inflammation, and tumor metastasis.(30,31) Below, we first consider the possible environmental and then the genetic factors impacting our patients’ dentoosseous phenotype. A) Non-Genetic Modifiers:

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Journal Pre-proof Neither of our patients had received medical or orthopedic treatment, and therefore we had encountered their disorder’s natural history. Vitamin D and iron deficiency in Patient 2, and limited dietary intake of Ca and Pi in both individuals, may have exacerbated their skeletal disease. Iron deficiency can increase FGF23 biosynthesis,(23) and indeed this phosphatonin circulated at a higher level in Patient 2. However, we were unable to learn more as both individuals were lost to follow-up after our studies.

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B) Heterozygous Unique CYP27B1 and ABCC6 Missense Mutations in Patient 1:

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Our propositus, whose dento-osseous disease seemed more severe compared to Patient 2,

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was older but additionally harbored heterozygous unique missense variants in two genes

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involved in mineral homeostasis, CYP27B1 and ABCC6. Although “carriers” of mutations in these genes are typically well, this finding merits brief discussion. CYP27B1, encoding the 1α-hydroxylase for converting 25(OH)D to

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1) CYP27B1:

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1,25(OH)2D, underlies AR vitamin D-dependent rickets, type IA (OMIM #264700).(3) Patient 1’s CYP27B1 missense variant (c.98C>T, Ala33Val) was not reported as a mutation in ClinVar,

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but nor as a SNP in ExAC. It predicted a conservative amino acid change with little impact on

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protein function. His biochemical testing did not include assay of circulating 1,25(OH)2D, but his mild hypocalcemia seemed attributable to the regional diet. 2) ABCC6:

ABCC6, an ATP-binding cassette transmembrane transporter gene, is

expressed primarily in the liver and kidney.(3) The encoded channel moves ATP extracellularly where its hydrolysis liberates the mineralization inhibitor inorganic pyrophosphate (PPi). In the two AR disorders caused by ABCC6 loss-of-function; GACI2 (OMIM #613312)(3) and PXE (OMIM # 264800)(3), ePPi deficiency leads to vascular calcification.(3,32,33)

Although

FGF23-mediated hypophosphatemia is not characteristic of either disorder, it can occur in

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Journal Pre-proof GACI1 featuring low ePPi from AR loss-of-function mutations of ectonucleotide pyrophosphatase 1 (ENPP1) that generates PPi from ATP.(3) PXE causes calcification of elastic fibers in skin, retina, and arteries.(3,32,33) Some heterozygous defects of ABCC6 lead to limited manifestations of PXE (Forme Fruste, OMIM # 177850)(3,34-36) including vascular calcification. Homozygous or compound heterozygous missense mutations near Patient 1’s heterozygous ABCC6 variant (e.g. Trp256Arg) have been reported in PXE. However, his unique heterozygous

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ABCC6 missense defect (Arg251Gln) was predicted to have little impact on the protein’s

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function despite encoding a non-conservative amino acid change, and he did not have PXE skin

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changes or vascular calcification on radiological imaging. We do not know if his diminished ePPi perhaps exacerbated his extreme enthesopathy. Enthesopathy is common in adults with (24)

featuring FGF23-mediated hypophosphatemia and, as we reported,(25) OPN

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XLH

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accumulates, as in Patient 1, around XLH osteocytes and within their canaliculi.

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C) DMP1 Deficiency:

It may be that DMP1 deficiency alone accounted for our patients’ dento-osseous disease.

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DMP1 is highly expressed in osteocytes. When lacking, there is altered Haversian canal/osteon

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microarchitecture,(37) and increased skeletal FGF23 secretion leading to hypophosphatemic rickets or osteomalacia.(3) In DMP1 deficient mice, the principal cellular defect may be altered osteoblast-to-osteocyte maturation leading to inappropriate expression of typical “osteoblastic” or “early osteocyte” genes, including those that encode type 1 collagen, ALP, and FGF23 produced by mature embedded osteocytes. In a rabbit DMP1 knockout model, accelerated chondrogenesis was noted(37) and therefore this type of disturbance perhaps contributed to our patients’ enthesopathy.

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Journal Pre-proof To assess whether our patients’ homozygous mutations affecting the two SIBLING proteins DMP1 and OPN caused dento-osseous disease more severe than ARHR1, we contrasted their heights to reports of ARHR1. The etiology of ARHR1 was discovered in 2006 in three families by Lorenz-Depiereux et al(4) and in two families by Feng et al.(5) Turan et al(8) in 2010 detailed the findings of three affected children. That same year, Makitie et al(7) delineated the clinical and radiographic features of affected adults. Beck-Nielsen in 2012(10) reported a treated

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child. From such reports, we understand that ARHR1 features short deformed long bones, joint

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pain, contractures, cranial hyperostosis, enthesopathy, and calcification of paraspinal ligaments

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sometimes completely immobilizing the spine. However, few reports contain sufficient clinical,

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biochemical, or radiographic detail to contrast with our two patients’ findings. Thus, height Z-scores seemed the best comparator mindful our patients were likely disadvantaged by lack of

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medical or orthopedic treatment. Patient 2, who was younger than patient 1, had especially

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severe scoliosis. Using data from the USA’s Centers for Disease Control and Prevention (CDC) for Americans, Patients 1 and 2 had height Z-scores of -4.4 and -6.6, respectively. However,

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height is less in Tamil Nadu, India, although slowly improving.(38) Currently for men ages 40-49

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years, it averages 163.8 (SD = 6.5) cm or 5 ft 5.5 in ( 2.6 in), and for women ages 20-29 years averages 153.1 (6.1) cm or 5 ft 0.3 in (2.4 in). Thus, our patients’ ethnic height Z-scores would be less aberrant at -2.9 and -5.4, respectively. Lorenz-Depiereux et al(4) reported the heights Zscores of five affected adults, some treated medically, ranging from -2.3 to -3.6. GannageYared(11) described two adults with height Z-scores of -1.3 and -1.9. Koshida et al(12) reported untreated consanguineous adult Japanese siblings with height Z-scores of -5.1 and -4.4. Makitie et al(7) in 2010 reported adult Finnish siblings whose height Z-scores were -5.1 and -6.4. Thus, the short statue of our two patients seemed in keeping with other adults with ARHR1, and we are

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Journal Pre-proof uncertain if added to the DMP1 defect causing ARHR1 their SPP1 mutation altering OPN had an effect of their disorder’s expressivity (see below). D) OPN Alteration: The evidence that our patients’ SPP1 mutation was deleterious for the encoded OPN included its low frequency (0.0009391) in ExAC, and its prediction of a non-conservative amino acid change (Leu to Phe) although SIFT(21) assessed it as “tolerated” and PolyPhen-2 as

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“benign”.(22) OPN is a 314 amino acid residue protein; 16 amino acids comprise its signal In healthy

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peptide and three (#159-161) represent an Arg-Gly-Asp cell attachment site.(1)

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individuals, OPN is highly phosphorylated on serine (Ser) and threonine (Thr) clusters comprising 3-5 amino acid residues each,(30) but to what degree varies among tissues, with milk

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containing an especially high 36 phosphoresidues.(30)

Phosphorylated OPN, and its

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phosphorylated peptides (including the ASARM peptide) generated by PHEX cleavage, potently

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inhibits mineralization.(39) In mice, deletion of Spp1 leads to hypermineralized bone(40) and reduced mechanical toughness.(41) In our patients, FGF23-mediated hypophosphatemia would

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importantly impair skeletal mineralization. For our propositus, however, we were unable to

collected.

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document osteomalacia or hypermineralization since his vertebral sample was decalcified when Nevertheless, his histological peri-osteocytic lesions and associated OPN

accumulation were reminiscent of the well-characterized lesions of XLH.(25) Our patients had elevated bone mass and high circulating ALP and FGF23 levels, predicting at least some degree of osteomalacia (osteoidosis), as well as unmineralized peri-osteocytic lesions where pliancy could affect mechanosensation leading to altered remodeling, although remodeling was indeed present as indicated by areas of bone showing frequent cement lines. Phosphorylated OPN might act in energy dissipation as well as in regulating biomineralization through its interactions

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Journal Pre-proof with Ca, other minerals and proteins, and its covalent crosslinking.

In fact, Sroga and

Vashishth(42) in 2018 correlated bone strength to the degree of phosphorylation of bone matrix proteins. OPN phosphorylation was shown to decrease with aging, such that ~30% of OPN phosphorylation was lost by the ninth decade-of-life, perhaps increasing bone fragility and fracture risk. Our patients’ mutation in SPP1 (c.796C>T, p.Leu266Phe) could disrupt OPN function in

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several ways. It predicted OPN alteration adjacent to a serine residue (Ser267) that can be

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phosphorylated. Hence, such a change could act directly by altering the protein’s structure at

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this site. Alternatively, possibly it compromises phosphorylation at a serine residue cluster that

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includes Ser267 and flanking Ser263 and Ser270, but with unknown consequences related to that specific site. Although, this Leu266Phe mutation corresponds to no known enzymatic cleavage

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site for OPN, it neighbors one (Arg244) cleaved by pro-protein convertase 5/6. Thus, our

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patients’ OPN cleavage could also be altered.(43) Finally, the SPP1 mutation might compromise crosslinking of OPN by transglutaminase 2, which occurs nearby at Gln248, Lys252, and

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Lys283, consequently diminishing its degradation (possibly by PHEX or other enzymes) with its

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accumulation then inhibiting mineralization and/or modulating mechanosensation signaling.(39,44) Although our patients’ DMP1 mutation and increased circulating FGF23 would cause hypophosphatemic rickets/osteomalacia (i.e., ARHR1), their SPP1 variant and OPN accumulation could be causing signaling abnormalities (including in osteocyte mechanosensing) contributing to their excessive bone mass with regions of osteomalacia and peri-osteocytic lesions as seen in XLH.(25) In XLH, despite hypophosphatemic osteomalacia from elevated circulating FGF23 together with accumulated OPN, diffusely elevated BMD with osteosclerosis and hyperostosis is common.(45)

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Journal Pre-proof E) Treatment: Patient 2, if confirmed to lack iron, might benefit from supplementation because this deficiency can increase circulating FGF23 levels and treatment has improved iron-deficient FGF23-mediated autosomal dominant hypophosphatemic rickets (ADHR; OMIM # 193100).(3) However, intravenous repletion of iron merits caution because some preparations can elevate circulating FGF23 levels and cause renal Pi wasting.

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Although some success has been reported, there is too little information to know whether

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ARHR1 responds to conventional medical therapy for FGF23-mediated hypophosphatemic

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skeletal disease using activated vitamin D together with frequent oral Pi supplementation, and particularly if the enthesopathy is altered. Now, FGF23-mediated hypophosphatemia in XLH

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can be treated using the human anti-FGF23 monoclonal antibody, burosumab.(46-48) Burosumab

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was approved multinationally in 2018 for patients 1 year-of-age or older with XLH, and clinical

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trials are underway using it for other FGF23-mediated hypophosphatemic disorders like tumorinduced osteomalacia or cutaneous hypophosphatemic skeletal disease.(49) No reports have

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assessed its utility for ARHR1. Treated or untreated, it will be important to follow patients with

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ARHR1 closely to understand not only their rickets or osteomalacia but also any ectopic mineralization including enthesopathy.

VI)

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