Search

Neoplastic transformation is a rare but serious complication of Paget's disease of bone (PDB), occurring in fewer than 1% of individuals with polyostotic disease. Their prognosis is poor, with less than 50% surviving 5 years. In 2016, the genetic alteration of giant cell tumor (GCT) complicating PDB was identified as a founder germline mutation (P937R) in the ZNF687 gene. However, the study population was exclusively of Italian descent, and patients of different ethnic origins were not studied. To fill this gap, herein we performed mutation analysis of ZNF687 in a GCT in the pelvis of a 45-year-old black American woman with polyostotic PDB. The P937R mutation in ZNF687 was found in her tumor but, as expected, the ancestral haplotype that characterizes the Italian GCT/PDB patients was not found. Furthermore, we identified two additional Italian GCT/PDB patients with this ZNF687 mutation, now constituting a cohort of 18 GCT/PDB cases, all harboring the identical mutation. We also searched for ZNF687 mutations in a unique collection of tumor tissues derived from Italian PDB patients, including 28 osteosarcomas (OS/PDB), 8 undifferentiated sarcomas (SRC/PDB), 1 fibrosarcoma (FS/PDB), and 1 chondrosarcoma (CS/PDB). We identified the P937R mutation in one SRC/PDB and a different ZNF687 mutation (R331W) in 1 of 28 pagetic osteosarcomas. Thus, whereas GCT/PDB pathogenesis globally seems to involve the P937R mutation in ZNF687, other neoplasms associated with PDB seem to be less related to mutations in this gene. Finally, we identified the G34W mutation in the H3F3A gene in the maxillary tumor masses of two PDB patients, defining them as conventional GCT rather than GCT/PDB. Thus, combined molecular analysis of H3F3A and ZNF687 is essential to clarify the origin and diagnosis of tumors in PDB. © 2020 American Society for Bone and Mineral Research.

Four heterozygous in-frame tandem duplications of different lengths in TNFRSF11A, the gene that encodes receptor activator of nuclear factor κB (RANK), constitutively activate RANK and lead to high turnover skeletal disease. Each duplication elongates the signal peptide of RANK. The 18-base pair (bp) duplication at position 84 (84dup18) causes familial expansile osteolysis (FEO), the 15-bp duplication at position 84 (84dup15) causes expansile skeletal hyperphosphatasia (ESH), the 12-bp duplication at position 90 (90dup12) causes panostotic expansile bone disease (PEBD), and the 27-bp duplication causes early-onset Paget’s disease of bone (PDB2). The severity of the associated skeletal disease seems inversely related to the duplication’s length. Additional 15- and 18-bp duplications of TNFRSF11A fit this pattern. Herein, we delineate the skeletal disease of a middle-aged man of Mexican descent who we found to harbor a novel 27-bp tandem duplication at position 77 (77dup27) of TNFRSF11A. His disorder shares features, particularly hand involvement, with the single Japanese (75dup27) and Chinese (78dup27) kindreds with PDB2 (PDB2(Jpn) and PDB2(Chn), respectively). However, his distinct hearing loss developed later in adulthood compared to the other 27-bp families. He reported no morbidities during childhood, but in his late 20s developed unexplained tooth loss, low-trauma fractures, post-operative hypercalcemia, and painless enlargement of his hands. Biochemical studies showed elevated serum alkaline phosphatase (ALP), bone-specific ALP, C-telopeptide, and osteocalcin consistent with rapid bone remodeling. Radiologic imaging revealed remarkably lucent bones with vertebral compression fractures, calvarial lucencies, and thinned long bone cortices. DXA showed extremely low bone mineral density. His disorder genetically and phenotypically fits best with PDB2 and can be called PDB2(Mex).

X-linked hypophosphatemia (XLH), the most prevalent heritable renal phosphate (Pi) wasting disorder, is caused by deactivating mutations of PHEX. Consequently, circulating phosphatonin FGF23 becomes elevated and hypophosphatemia in affected children leads to rickets with skeletal deformity and reduced linear growth while affected adults suffer from osteomalacia and forms of ectopic mineralization. In 2015, we reported uniquely mild XLH in six children and four of their mothers carrying the non-coding PHEX 3'-UTR mutation c.*231A>G. Herein, we characterize this mild XLH variant by comparing its features in 30 individuals to 30 age- and sex-matched patients with XLH but without the 3'-UTR mutation. The "UTR" and "XLH" groups, both comprising 17 children (2 to 17 years, 3 girls) and 13 adults (23 to 63 years, 10 women), had mean ages of 23 years. Only 43% of the UTR group versus 90% of the XLH group had received medical treatment for their disorder, including 0% versus 85% of the females, respectively (ps < .0001). The UTR group was taller: mean ± SD height Z-score (HZ) -1.0 ± 1.0 versus -2.0 ± 1.4 (p = .0034), with significantly greater height for females (-0.9 ± 0.7 versus -2.3 ± 1.4; p = .0050) but not males (-1.2 ± 1.1 versus -1.9 ± 1.5; p = .1541), respectively. Mean ± SD "arm span Z-score" (AZ) did not differ between the UTR -0.8 ± 1.3 versus XLH -1.3 ± 1.8 groups (p = .2269). Consequently, the UTR group was more proportionate with a mean ∆Z (AZ - HZ) of 0.1 ± 0.6 versus 0.7 ± 1.0 (p = .0158), respectively. Compared to the XLH group, the UTR group had significantly higher fasting serum Pi and renal tubular threshold maximum for phosphorus per glomerular filtration rate (TmP/GFR) (ps ≤ .0060), serum FGF23 concentrations within the reference range (p = .0068), and similar serum alkaline phosphatase levels (p = .6513). UTR lumbar spine bone mineral density Z-score was higher (p = .0343). Thus, the 3'-UTR variant of XLH is distinctly mild, especially in girls and women, posing challenges for its recognition and management. © 2020 American Society for Bone and Mineral Research.

Background: Cutaneous skeletal hypophosphatemia syndrome (CSHS) is caused by somatic mosaicism of a RAS family gene (ie. HRAS, NRAS, KRAS). CSHS features: 1) congenital epidermal, melanocytic, or sebaceous nevi, 2) elevated circulating FGF23 levels that cause renal phosphate wasting and skeletal hypomineralization, and 3) focal bone lesions ipsilateral to the nevi as a possible source of excess FGF23. Conventional therapy for rickets in CSHS (phosphate salts and bioactive vitamin D) mirrors treatment for X-linked hypophosphatemia (XLH) with elevated circulating FGF23. Current therapy aims to treat signs and symptoms rather than the etiology. Clinical Case: A 21-month-old boy with left, widespread, unilateral nevus sebaceous developed bilateral lower extremity bowing with a wide-based gait. His serum phosphorus level was low at 2.1 mg/dl (Nl: 3-6) and FGF23 was elevated at 279 RU/mL (Nl: < 230). We prescribed conventional therapy for hypophosphatemic rickets and diagnosed linear sebaceous nevus syndrome, confirmed by mutation analysis of affected skin harboring HRAS p.Q61R (c.182A>G). Adherence to medical therapy was problematic, and he sustained 3 major long-bone fractures. By age 6 years, a walker was required to ambulate short distances with mostly wheelchair-dependence from pain and muscle weakness. Based on his elevated FGF23 level and clinical decline, burosumab (anti-FGF23 fully human monoclonal antibody), FDA-approved in 2018 for XLH, was initiated off-label. After weaning conventional therapy, burosumab was administered subcutaneously as a single dose of 0.8 mg/kg (20 mg) and decreased to 0.3 mg/kg (6 mg) two weeks later. Burosumab was then given every 2 weeks and adjusted by 0.1 mg/kg to maintain fasting serum phosphorus in the low-normal range for age (3.5 - 4.5 mg/dL). Laboratory and clinic follow-up occurred just prior to each injection for 3 months until serum phosphorus was stable as he received approximately 0.4 mg/kg (8 mg per dose). Biochemical testing and clinic visits will occur monthly for 3 months and are planned for quarterly afterwards, with repeat radiological imaging every 3-6 months for the first year of therapy. Duration of burosumab treatment has been 5 months. After his initial burosumab dose, serum phosphorus increased from 1.7 to 3.7 mg/dL and remained normal [Nl: 3.0 - 6.0 mg/dL] thereafter. Radiographs of his wrists and knees 3 months into therapy revealed striking improvement of his rickets but persisting focal bone lesions, characteristic of CSHS, on the left side. He now walks independently with endurance, strength, and resolving pain. Quality-of-life is markedly improved. Burosumab treatment is being tolerated well without side effects. Conclusion: Burosumab seems to be an effective therapy for FGF23-mediated rickets in pediatric CSHS. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Background: Skeletal fluorosis (SF) is endemic in many places, especially where well water is rich in fluoride (F) from volcanic rock. SF is rare in the US, where it has unusual causes. The impact of F on the skeleton is conditioned by calcium and vitamin D sufficiency. Clinical Case: A 51-year-old obese man with chronic opiate use was referred for secondary hyperparathyroidism detected after right femoral neck and left proximal femur fractures, and displaced humeral fracture which healed poorly with radial nerve entrapment. Oncologic evaluation was negative, including intraoperative bone biopsy. He reported longstanding diffuse musculoskeletal pain, drank 72 oz of cola daily, and consumed little dietary calcium. Physical exam showed Ht 1.7 m, BMI 46, no dental abnormalities, deformed right humerus, diminished right wrist dorsiflexion, and an antalgic gait. DXA BMD Z-score was +7.4 at the spine and +0.4 at the “1/3” radius. At femur fracture, corrected serum calcium was 7.8 mg/dL (8.5-10.1) and alkaline phosphatase (ALP) 1080 U/L (46-116). After 5 months of calcium and vitamin D supplementation, calcium was 9.4 mg/dL, ALP 539 U/L, phosphorus 3.7 mg/dL (2.3-4.7), 25(OH)D 20.6 ng/mL (30-100), PTH 327 pg/mL (8.7-77.1), and creatinine 0.62 mg/dL (0.72-1.25). Hepatitis C Ab and PSA were normal. Elevated serum C-telopeptide 2513 pg/mL (87-345) and osteocalcin greater than 300 ng/mL (9-38) indicated rapid bone turnover. Bone scan showed increased uptake at the left hip fracture site, 2 ribs, and periarticular areas. Radiographic skeletal survey revealed diffuse osteosclerosis. Mutation analysis for high turnover sclerotic skeletal disease was negative, including examination of OPG, exon 1 of RANK and a NSG high bone mass panel. Initially, F exposure history was negative, however serum and urine F levels were elevated at 118 mcmol/L (0-4) and 42.6 mg/L (0.3-3.2), respectively. His mother confided that he had “huffed” difluoroethane containing computer cleaner for 2 years several times daily to control pain. Subsequently, right femur fracture required intramedullary fixation, several weeks rehabilitation, and generous doses of calcium and vitamin D3. Routine nondecalcified histology showed reactive bone with reticulin and collagen fibrosis. 24-hour urine of 3.43 liters contained calcium less than 68.6 mg and F 91.6 mg (0.2-3.2 mg/L). Conclusion: Inhalant abuse of fluorocarbons is known. However, literature concerning the skeletal effects is scant. Our patient’s positive bone balance together with low calcium intake could explain his secondary hyperparathyroidism. High phosphorus in colas may also have decreased gastrointestinal calcium absorption. Deposition of fluorohydroxyapatite in the skeleton likely explained our patient’s skeletal fragility.

XLH is characterized by excess FGF23, hypophosphatemia, skeletal deformities, and growth impairment. For the last 40 years, XLH has been treated with multiple daily doses of oral phosphate and active vitamin D (Pi/D). Burosumab, a fully human monoclonal antibody to FGF23, has been approved by the FDA for the treatment of XLH in patients ≥1 year-old. In this Phase 3 trial (NCT02915705), 61 children with XLH (1-12 years old) were randomized (1:1) to receive subcutaneous burosumab starting at 0.8 mg/kg every 2 weeks or continue Pi/D titrated and individualized for each subject by investigators. Eligibility criteria included a Total Rickets Severity Score (RSS) ≥2.0 despite prior treatment with Pi/D (>7-day washout before baseline). The primary endpoint was healing of rickets at Week 40 assessed by radiologists blinded to treatment using the Radiographic Global Impression of Change (RGI-C). The mean ± SE daily oral phosphate dose from baseline to Week 40 was 37.8 ± 3.2 mg/kg, with >99% compliance reported based on days of dosing. Compared with Pi/D, 40 weeks of burosumab resulted in a greater LS mean ± SE increase in serum phosphorus (0.92 ± 0.08 vs 0.20 ± 0.06 mg/dL), TmP/GFR (1.19 ± 0.11 vs -0.16 ± 0.05 mg/dL), and 1,25(OH)2D (30 ± 4 vs 19 ± 4 pg/mL). At Week 40, rickets improved in both groups; RGI-C global score was significantly higher in burosumab subjects than in Pi/D subjects (LS mean ± SE: +1.9 ± 0.1 vs +0.8 ± 0.1; p

Asfotase alfa (AA), an enzyme replacement therapy, is the only approved medical treatment for pediatric-onset hypophosphatasia (HPP). We report the safety profile of AA in patients from the clinical trial program. Safety data were pooled from 4 open-label, multicenter studies in children age ≤3 years (study 002/003 [NCT00744042/NCT01205152]; n=11) and age ≤5 years (study 010-10 [NCT01176266]; n=69) with onset of HPP symptoms before age 6 months, children age 5-12 years with HPP and open growth plates (study 006/008 [NCT00952484/NCT01203826]; n=13), and adolescents and adults age 13-65 years with onset of HPP at any age (study 009 [NCT01163149]; n=19). AA doses varied between studies; data were available for up to 7 years from studies 002/003, 010-10, and 006/008 and for up to 5 years from study 009. Safety events after the studies ended were not included. In total, data were available for 112 patients (median [min, max] age at enrollment: 3.2 [0, 66.5] y; female: 51%; white: 84%). Median (min, max) treatment duration was 2.7 years (1 d, 7.5 y). The average weekly total dose ranged from 2.1 to 11.9 mg/kg. All patients experienced adverse events (AEs); 26% of events were considered related to treatment and were mild (74%) or moderate (21%) in severity. Serious treatment-related AEs were reported in 10 (9%) patients. Serious AEs of special interest were craniosynostosis (25%; including increased intracranial pressure), injection-associated reactions (5%; including hypersensitivity), injection site reactions (ISRs) (2%; including erythema), ectopic calcifications (2%; including nephrolithiasis), and liver abnormalities or disease (2%; including chronic hepatitis). Overall, ISRs were the most common treatment-related AEs (73%). Ten deaths occurred among patients with severe HPP who entered the studies as infants (1 day to 20 months). Four of these deaths were attributed to pneumonia/sepsis, and 1 death each was attributed to respiratory failure and cerebral death; HPP-related complications; severe respiratory failure; cardiopulmonary arrest; severe cardiopulmonary insufficiency; and transtentorial and cerebellar tonsillar herniation due to cerebral edema. The majority of deaths were considered related to underlying HPP; one of the deaths, attributed to pneumonia, was reported by the investigator as being possibly related to AA. AEs leading to AA withdrawal were reported in 11 (10%) patients; those reported in ≥2 patients included pneumonia and respiratory failure. Anti-AA antibody data were available for 109 patients; 97 (89%) tested positive, and 55 (57%) of these showed presence of neutralizing antibodies at some time during the studies. In this pooled analysis of mostly prepubescent children (84%) treated with AA for up to 7 years, ISRs were the most frequently reported treatment-related AEs. Funding: This study was sponsored by Alexion Pharmaceuticals, Inc.

LRP5 encodes low-density lipoprotein receptor-related protein 5 (LRP5). When LRP5 with a Frizzled receptor join on the surface of an osteoblast and bind a member of the Wnt family of ligands, canonical Wnt/β-catenin signaling occurs and increases bone formation. Eleven heterozygous gain-of-function missense mutations within LRP5 are known to prevent the LRP5 inhibitory ligands sclerostin and dickkopf1 from attaching to LRP5's first β-propeller, and thereby explain the rare autosomal dominant (AD) skeletal disorder "high bone mass" (HBM). LRP6 is a cognate co-receptor of LRP5 and similarly controls Wnt signaling in osteoblasts, yet the consequences of increased LRP6-mediated signaling remain unknown. We investigated two multi-generational American families manifesting the clinical and routine laboratory features of LRP5 HBM but without an LRP5 defect and instead carrying a heterozygous LRP6 missense mutation that would alter the first β-propeller of LRP6. In Family 1 LRP6 c.602CT, p.A201V was homologous to LRP5 HBM mutation c.641CT, p.A214V, and in Family 2 LRP6 c.553AC, p.N185H was homologous to LRP5 HBM mutation c.593AG, p.N198S but predicting a different residue at the identical amino acid position. In both families the LRP6 mutation co-segregated with striking generalized osteosclerosis and hyperostosis. Clinical features shared by the seven LRP6 HBM family members and ten LRP5 HBM patients included a broad jaw, torus palatinus, teeth encased in bone and, reportedly, resistance to fracturing and inability to float in water. For both HBM disorders, all affected individuals were taller than average for Americans (Ps 0.005), but with similar mean height Z-scores (P = 0.7606) and indistinguishable radiographic skeletal features. Absence of adult maxillary lateral incisors was reported by some LRP6 HBM individuals. In contrast, our 16 patients with AD osteopetrosis [i.e., Albers-Schönberg disease (A-SD)] had an unremarkable mean height Z-score (P = 0.9401) lower than for either HBM group (Ps 0.05). DXA mean BMD Z-scores in LRP6 HBM versus LRP5 HBM were somewhat higher at the lumbar spine (+7.8 vs +6.5, respectively; P = 0.0403), but no different at the total hip (+7.9 vs +7.7, respectively; P = 0.7905). Among the three diagnostic groups, only the LRP6 HBM DXA BMD values at the spine seemed to increase with subject age (R = +0.7183, P = 0.0448). Total hip BMD Z-scores were not significantly different among the three disorders (Ps 0.05), and showed no age effect (Ps 0.1). HR-pQCT available only for LRP6 HBM revealed indistinct corticomedullary boundaries, high distal forearm and tibial total volumetric BMD, and finite element analysis predicted marked fracture resistance. Hence, we have discovered mutations of LRP6 that cause a dento-osseous disorder indistinguishable without mutation analysis from LRP5 HBM. LRP6 HBM seems associated with generally good health, providing some reassurance for the development of anabolic treatments aimed to enhance LRP5/LRP6-mediated osteogenesis.

XLH is the most common heritable rickets. Affected children have high levels of circulating FGF23 that cause hypophosphatemia with consequent rickets and lower limb deformity. Burosumab, a fully human monoclonal antibody that binds FGF23, is FDA-approved for the treatment of XLH in children ≥6 months old and adults. Herein, we report final, 3-year safety and efficacy data from an open-label, phase 2 study of burosumab in children 1 to Eligibility required hypophosphatemia and radiographic evidence of rickets. The primary efficacy endpoint was change from baseline in fasting serum phosphorus (Pi). Secondary endpoints included Rickets Severity Score (RSS) and Radiographic Global Impression of Change (RGI-C). Patients received burosumab subcutaneous Q2W starting at 0.8 mg/kg for 160 weeks (64-week treatment + 96-week treatment extension periods). All 13 enrolled patients completed the 64-week treatment period; 1 left the study to transition to commercially available burosumab, and 12 completed all 160 weeks. Baseline mean (SD) age was 2.9 (1.1) years; 69% were boys; all had previously received oral phosphate salts and active vitamin D. Burosumab rapidly corrected fasting serum Pi with mean (SD) levels of 2.5 (0.3) mg/dL at Baseline, 3.7 (0.5) mg/dL at Week 1 (W1), 3.4 (0.5) mg/dL at W64, and 3.4 (0.5) mg/dL at W160 (normal range: 3.2–6.1 mg/dL). Lower RSS indicated improved rickets. Total RSS decreased from 2.9 (1.4) at Baseline to 1.2 (0.5) at W40 and to 0.9 (0.5) at W64 and was maintained through W160 [1.0 (0.6)]. Positive RGI-C scores indicate healing rickets relative to Baseline. Global RGI-C scores indicating substantial healing (≥+2) at W40 [+2.2 (0.3)] and W64 [+2.2 (0.4)] were maintained through W160 [+2.2 (0.4)]. Similarly, lower limb deformity RGI-C scores were +1.2 (0.6) at W40 and +1.5 (0.5) at W64, and sustained healing was evident at W160 [+2.0 (0.3)]. Wrist and knee RSSs and RGI-C scores similarly improved. The upper limit of normal for serum ALP ranged from 297 to 345 U/L depending on the child’s age and sex. Mean ALP was 549 (194) U/L at Baseline, normalized by W40 [335 (88) U/L], and was sustained through W160 [302 (71) U/L]. The burosumab safety profile over 160 weeks resembled previous pediatric studies; no new safety concerns emerged. All patients had ≥1 treatment-emergent adverse event (TEAE). All TEAEs were mild (Grade 1) or moderate (Grade 2) except for one patient with a grade 3 TEAE (food allergy) and one with a grade 3 TEAE (increased serum amylase, 92% salivary/8% pancreatic). One patient had a serious TEAE (dental abscess leading to hospitalization). These grade 3 and serious TEAEs were considered unrelated to study drug. Burosumab rapidly restored Pi homeostasis, improved rickets, and normalized serum ALP in children with XLH aged 1 to

Skeletal fluorosis (SF) is endemic primarily in regions with fluoride (F)-contaminated well water, but can reflect other types of chronic F exposure. Calcium (Ca) and vitamin D (D) deficiency can exacerbate SF. A 51-year-old man with years of musculoskeletal pain and opiate use was hypocalcemic with secondary hyperparathyroidism upon manifesting recurrent long bone fractures. He smoked cigarettes, drank large amounts of cola beverage, and consumed little dietary Ca. Then, after 5 months of Ca and D(3) supplementation, serum 25(OH)D was 21 ng/mL (Nl, 30–100), corrected serum Ca had normalized from 7.8 to 9.4 mg/dL (Nl, 8.5–10.1), alkaline phosphatase (ALP) had decreased from 1080 to 539 U/L (Nl, 46–116), yet parathyroid hormone (PTH) had increased from 133 to 327 pg/mL (Nl, 8.7–77.1). Radiographs revealed generalized osteosclerosis and a cystic osteopenic area in the left femoral neck and intertrochanteric region. DXA BMD Z-scores were +7.4 and +0.4 at the lumbar spine and “1/3” radius, respectively. Bone scintigraphy showed increased uptake in two ribs, periarticular areas, and proximal left femur at the site of a subsequent atraumatic fracture. Elevated serum collagen type I C-telopeptide 2513 pg/mL (Nl, 87–345) and osteocalcin >300 ng/mL (Nl, 9–38) indicated rapid bone turnover. Negative studies included hepatitis C Ab, prostate-specific antigen, serum and urine electrophoresis, and Ion Torrent mutation analysis for dense or high-turnover skeletal diseases. After discovering markedly elevated F concentrations in his plasma [4.84 mg/L (Nl, 0.02–0.08)] and spot urine [42.6 mg/L (Nl, 0.2–3.2)], a two-year history emerged of “huffing” computer cleaner containing difluoroethane. Non-decalcified histology of a subsequent right femur fracture showed increased osteoblasts and osteoclasts and excessive osteoid. A 24-hour urine collection contained 27 mg/L F (Nl, 0.2–3.2) and < 2 mg/dL Ca. Then, 19 months after “huffing” cessation and improved Ca and D(3) intake, yet with persisting bone pain, serum PTH was normal (52 pg/mL) and serum ALP and urine F had decreased to 248 U/L and 3.3 mg/L, respectively. Our experience combined with 15 publications in PubMed concerning unusual causes of non-endemic SF where the F source became known (19 cases in all) revealed: 11 instances from high consumption of black tea and/or F-containing toothpaste, 1 due to geophagia of F-rich soil, and 7 due to “recreational” inhalation of F-containing vapors. Circulating PTH measured in 13 was substantially elevated in 2 (including ours) and mildly increased in 2. Their SF severity, including bone turnover rate, seemed to reflect cumulative F exposure, renal function, and Ca and D status. Several factors appeared to condition our patient’s skeletal disease: i) direct anabolic effects of toxic amounts of F on his skeleton, ii) secondary hyperparathyroidism from degradation-resistant fluorapatite bone crystals and low dietary Ca, and iii) impaired mineralization of excessive osteoid due to hypocalcemia.

Hypophosphatasia (HPP) is caused by loss-of-function mutation(s) of the gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Autosomal inheritance (dominant or recessive) from among more than 300 predominantly missense defects of TNSALP (ALPL) explains HPP's broad-ranging severity, the greatest of all skeletal diseases. In health, TNSALP is linked to cell surfaces and richly expressed in the skeleton and developing teeth. In HPP,TNSALP substrates accumulate extracellularly, including inorganic pyrophosphate (PPi), an inhibitor of mineralization. The PPi excess can cause tooth loss, rickets or osteomalacia, calcific arthropathies, and perhaps muscle weakness. Severely affected infants may seize from insufficient hydrolysis of pyridoxal 5'-phosphate (PLP), the major extracellular vitamin B6 . Now, significant successes are documented for newborns, infants, and children severely affected by HPP given asfotase alfa, a hydroxyapatite-targeted recombinant TNSALP. Since fall 2015, this biologic is approved by regulatory agencies multinationally typically for pediatric-onset HPP. Safe and effective treatment is now possible for this last rickets to have a medical therapy, but a number of challenges involving diagnosis, understanding prognosis, and providing this treatment are reviewed herein. © 2017 American Society for Bone and Mineral Research.

In 1985, we briefly reported infant sisters with a unique, lethal, autosomal recessive disorder designated congenital sclerosing osteomalacia with cerebral calcification. In 1986, this condition was entered into Mendelian Inheritance In Man (MIM) as osteomalacia, sclerosing, with cerebral calcification (MIM 259660). However, no attestations followed. Instead, in 1989 Raine and colleagues published an affected neonate considering unprecedented the striking clinical and radiographic features. In 1992, “Raine syndrome” entered MIM formally as osteosclerotic bone dysplasia, lethal (MIM #259775). In 2007, the etiology emerged as loss-of-function mutation of FAM20C that encodes family with sequence similarity 20, member C. FAM20C is highly expressed in embryonic calcified tissues and encodes a kinase (dentin matrix protein 4) for most of the secreted phosphoproteome including FGF23, osteopontin, and other regulators of skeletal mineralization. Herein, we detail the clinical, radiological, biochemical, histopathological, and FAM20C findings of our patients. Following premortem tetracycline labeling, the proposita's non-decalcified skeletal histopathology after autopsy indicated no rickets but documented severe osteomalacia. Archival DNA revealed the sisters were compound heterozygotes for a unique missense mutation and a novel deletion in FAM20C. Individuals heterozygous for the missense mutation seemed to prematurely fuse their metopic suture and develop a metopic ridge sometimes including trigonocephaly. Our findings clarify FAM20C's role in hard tissue formation and mineralization, and show that Raine syndrome is congenital sclerosing osteomalacia with cerebral calcification. © 2016 American Society for Bone and Mineral Research.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism that features deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Resultant extracellular accumulation of inorganic pyrophosphate, a TNSALP substrate and potent inhibitor of mineralization, typically leads to tooth loss and sometimes to rickets or osteomalacia. HPP's remarkably broad-ranging severity is largely explained by autosomal dominant versus autosomal recessive transmission from among several hundred usually missense mutations positioned throughout the gene that encodes TNSALP. In 2015, our cross-sectional investigation of 173 affected children validated and expanded the clinical nosology commonly used for pediatric HPP. Herein, for the 101 patients in that cohort with longitudinal data, we explored the natural history of pediatric HPP by assessing their z-scores for height and then for weight, grip strength, and bone mineral density (BMD) determined by dual energy X-ray absorptiometry (DXA) also after adjusting for patient height. Eighteen patients contributed to "across" puberty evaluation. According to increasing HPP severity, there were 28 odonto HPP, 28 mild childhood HPP, 37 severe childhood HPP, and 8 infantile HPP patients typically studied from early to mid-childhood. The individual values for each parameter were wide-ranging within, and overlapping between, the four successive patient groups. Final mean/median z-scores, like the published initial values, paralleled the nosology. Longitudinal findings were similar for the boys versus girls and across puberty. Mean/median height z-scores remained constant for all four patient groups. In contrast, mean/median weight z-scores increased with aging, including after height-adjustment, resembling the recent trend for American children. However, excessive weight gain was typically not observed and mean/median values became average for height. Mean/median z-scores calculated routinely for chronologic age did not change for grip strength or for lumbar spine or total hip BMD. However, height-correction of the cohort suggested some worsening of grip strength z-scores and indicated improvement in spine BMD z-scores. Overall, in affected children and adolescents, HPP represents a clinically stable but chronic disorder.

Hypophosphatasia is the inborn error of metabolism characterized by low serum alkaline phosphatase activity (hypophosphatasaemia). This biochemical hallmark reflects loss-of-function mutations within the gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP is a cell-surface homodimeric phosphohydrolase that is richly expressed in the skeleton, liver, kidney and developing teeth. In hypophosphatasia, extracellular accumulation of TNSALP natural substrates includes inorganic pyrophosphate, an inhibitor of mineralization, which explains the dento-osseous and arthritic complications featuring tooth loss, rickets or osteomalacia, and calcific arthopathies. Severely affected infants sometimes also have hypercalcaemia and hyperphosphataemia due to the blocked entry of minerals into the skeleton, and pyridoxine-dependent seizures from insufficient extracellular hydrolysis of pyridoxal 5'-phosphate, the major circulating form of vitamin B6, required for neurotransmitter synthesis. Autosomal recessive or dominant inheritance from ~300 predominantly missense ALPL (also known as TNSALP) mutations largely accounts for the remarkably broad-ranging expressivity of hypophosphatasia. High serum concentrations of pyridoxal 5'-phosphate represent a sensitive and specific biochemical marker for hypophosphatasia. Also, phosphoethanolamine levels are usually elevated in serum and urine, though less reliably for diagnosis. TNSALP mutation detection is important for recurrence risk assessment and prenatal diagnosis. Diagnosing paediatric hypophosphatasia is aided by pathognomic radiographic changes when the skeletal disease is severe. Hypophosphatasia was the last type of rickets or osteomalacia to await a medical treatment. Now, significant successes for severely affected paediatric patients are recognized using asfotase alfa, a bone-targeted recombinant TNSALP.

Alkaline phosphatase can be considered "our favorite enzyme" for reasons apparent to those who diagnose and treat metabolic bone diseases or who study skeletal biology. Few might know, however, that alkaline phosphatase likely represents the most frequently assayed enzyme in all of medicine. Elevated activity in the circulation is universally recognized as a marker for skeletal or hepatobiliary disease. Nevertheless, the assay conditions in many ways are nonphysiological. The term alkaline phosphatase emerged when it became necessary to distinguish "bone phosphatase" from the phosphatase in the prostate that features an acidic pH optimum. Beginning in 1948, studies of the inborn-error-of-metabolism hypophosphatasia would identify the natural substrates and establish the physiological role of alkaline phosphatase, including in biomineralization. Here, we recount the discovery in 1923 and then eventual naming of this enzyme that remains paramount in our field. © 2017 American Society for Bone and Mineral Research.

Wnt/β-catenin signaling is important for skeletal development and health. Eleven heterozygous gain-of-function missense mutations within the first β-propeller of low-density lipoprotein receptor-related protein 5 (LRP5) are known to cause the autosomal dominant disorder called high bone mass (HBM). In 2019, different heterozygous LRP6 missense mutations were identified in two American families with the HBM phenotype but including absent lateral maxillary and mandibular incisors. We report a 19-year-old Argentinian man referred for "osteopetrosis" and nine years of generalized, medium-intensity bone pain and arthralgias of both knees. His jaw and nasal bridge were broad and several teeth were missing. Routine biochemical testing, including of mineral homeostasis, was normal. Urinary deoxypyridinoline and serum CTX were slightly increased. Radiographic skeletal survey showed diffusely increased radiodensity. DXA revealed substantially elevated BMD Z-scores. Digital orthopantomography confirmed agenesis of his maxillary and mandibular lateral incisors and his second left superior premolar. Cranial magnetic resonance imaging showed diffuse thickening of the calvarium and skull base, dilation of the sheath of the optic nerves containing increased fluid and associated with subtle stenosis of the optic canal, and narrow internal auditory canals. Mutation analyses identified a heterozygous indel mutation in exon 4 of LRP6 involving a single nucleotide change and 6-nucleotide deletion (c.678T>Adel679-684, p.His226Gln-del227-228ProPhe) leading to a missense change and 2-amino acid deletion that would compromise the first β-propeller of LRP6. Experience to date indicates LRP6 HBM is indistinguishable from LRP5 HBM without mutation analysis, although in LRP6 HBM absence of adult lateral incisors may prove to be a unique feature.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP in healthy individuals is on cell surfaces richly in bone, liver, and kidney. Thus, TNSALP natural substrates accumulate extracellularly in HPP, including inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite crystal formation and growth. Superabundance of extracellular PPi (ePPi) in HPP impairs mineralization of bones and teeth, often leading to rickets during childhood and osteomalacia in adult life and to tooth loss at any age. HPP’s remarkably broad-ranging severity is largely explained by nearly four hundred typically missense mutations throughout the ALPL gene that are transmitted as an autosomal dominant or autosomal recessive trait. In the clinical laboratory, the biochemical hallmark of HPP is low serum ALP activity (hypophosphatasemia). However, our experience indicates that hyperphosphatemia from increased renal reclamation of filtered inorganic phosphate (Pi) is also common. Herein, from our prospective single-center study, we document throughout the clinical spectrum of non-lethal pediatric HPP that hyperphosphatemia reflects increased renal tubular threshold maximum for phosphorus adjusted for the glomerular filtration rate (TmP/GFR). To explore its pathogenesis, we studied mineral metabolism and quantitated circulating levels of three phosphatonins [fibroblast growth factor 23 (FGF23), secreted frizzled-related protein 4 (sFRP4), and fibroblast growth factor 7 (FGF7)] in 41 pediatric patients with HPP, 73 with X-linked hypophosphatemia (XLH), and 15 healthy pediatric control (CTR) subjects. The HPP and XLH cohorts had normal serum total and ionized calcium and parathyroid hormone levels (Ps > 0.10) and uncompromised glomerular filtration. In XLH, serum FGF23 was characteristically elevated (P < 0.0001) and despite hypophosphatemia sFRP4 was normal (P > 0.4) while FGF7 was low (P < 0.0001). In HPP, despite hyperphosphatemia serum FGF23 and sFRP4 were normal (Ps > 0.8) while FGF7 was low (P < 0.0001). Subsequently, in rats, we confirmed that FGF7 is phosphaturic. Thus, hyperphosphatemia in non-lethal pediatric HPP is associated with phosphatonin insufficiency together with, as we discuss, ePPi excess and diminished renal TNSALP activity.

Metabolic bone diseases comprise a diverse group of disorders characterized by alterations in skeletal homeostasis, and are often associated with abnormal circulating concentrations of calcium, phosphate or vitamin D metabolites. These diseases commonly have a genetic basis and represent either a monogenic disorder due to a germline or somatic single gene mutation, or an oligogenic or polygenic disorder that involves variants in more than one gene. Germline single gene mutations causing Mendelian diseases typically have a high penetrance, whereas the genetic variations causing oligogenic or polygenic disorders are each associated with smaller effects with additional contributions from environmental factors. Recognition of familial monogenic disorders is of clinical importance to facilitate timely investigations and management of the patient and any affected relatives. The diagnosis of monogenic metabolic bone disease requires careful clinical evaluation of the large diversity of symptoms and signs associated with these disorders. Thus, the clinician must pursue a systematic approach beginning with a detailed history and physical examination, followed by appropriate laboratory and skeletal imaging evaluations. Finally, the clinician must understand the increasing number and complexity of molecular genetic tests available to ensure their appropriate use and interpretation.

Objective: To report clinical characteristics and medical history data obtained retrospectively for a large cohort of pediatric patients with perinatal and infantile hypophosphatasia. Study design: Medical records from academic medical centers known to diagnose and/or treat hypophosphatasia were reviewed. Patients born between 1970 and 2011 with hypophosphatasia and any of the following signs/symptoms at age 70% of patients between birth and age 5 years. Vitamin B6–dependent seizures and respiratory distress and failure were associated significantly (P

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 least one “acidic, serine- and aspartic acid-rich motif’ (ASARM) and multiple Ser-x-Glu/pSer sequences that when phosphorylated promote binding of the protein to hydroxyapatite for regulation of biomineralization. Mendelian disorders from loss-of-function mutation(s) of the genes that encode the SIBLINGs thus far involve DSPP causing various autosomal dominant 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-function mutation(s) of DSPP or DMP1 or from alterations of SPP1, IBSP, or MEPE. Herein, we describe severe hypophosphatemic osteosclerosis and hyperostosis associated with skeletal deformity, short stature, enthesopathy, tooth loss, and high circulating FGF23 levels 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 surrounding osteocytes. We review how our patients’ disorder may represent the first digenic SIBLING protein osteopathy.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism characterized enzymatically by insufficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and caused by either mono- or bi-allelic loss-of-function mutation(s) of the gene ALPL that encodes this cell surface phosphomonoester phosphohydrolase. In HPP, the natural substrates of TNSALP accumulate extracellularly and include inorganic pyrophosphate (PPi), a potent inhibitor of biomineralization. This PPi excess leads to rickets or osteomalacia in all but the most mild “odonto” form of the disease. Adults with HPP understandably often also manifest calcium PPi dihydrate deposition, whereas enthesopathy and calcific periarthritis from hydroxyapatite (HA) crystal deposition can seem paradoxical in face of the defective skeletal mineralization. In 2015, asfotase alfa (AA), a HA-targeted TNSALP, was approved multinationally as an enzyme replacement therapy for HPP. AA hydrolyzes extracellular PPi (ePPi) and in HPP enables HA crystals to grow and mineralize skeletal matrix. In direct contrast to HPP, deficiency of ePPi characterizes the inborn-errors-of-metabolism generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE). In GACI and PXE, deficiency of ePPi leads to ectopic mineralization including vascular calcification (VC). Therefore, in HPP, ectopic mineralization including VC could hypothetically result from, or be exacerbated by, the persistently high circulating TNSALP activity that occurs during AA treatment. Herein, using a routine computed tomography (CT) method to quantitate coronary artery calcium, we found no ectopic mineralization in the heart of an elderly woman with HPP before or after 8 months of AA treatment. Subsequently, investigational high-resolution peripheral quantitative CT and dual-energy X-ray absorptiometry showed absence of peripheral artery and aortic calcium after further AA treatment. Investigation of additional adults with HPP could reveal if the superabundance of ePPi protects against VC, and whether long-term AA therapy causes or exacerbates any ectopic mineralization.

Children with X-linked hypophosphataemia have high concentrations of circulating phosphatonin fibroblast growth factor 23 (FGF23), which causes renal phosphate wasting and hypophosphataemia, rickets, skeletal deformities, and growth impairment. Burosumab, a human monoclonal antibody against FGF23, improves phosphate homoeostasis and rickets in children aged 5-12 years with X-linked hypophosphataemia. We aimed to assess the safety and efficacy of burosumab in younger children with X-linked hypophosphataemia.In this open-label, phase 2 trial at three hospitals in the USA, children (aged 1-4 years) with X-linked hypophosphataemia received burosumab (0·8 mg/kg) via subcutaneous injection every 2 weeks for 64 weeks. The dose was increased to 1·2 mg/kg if two consecutive pre-dose serum phosphorus concentrations were below 1·03 mmol/L (3·2 mg/dL), serum phosphorus had increased by less than 0·16 mmol/L (0·5 mg/dL) from baseline, and a dose of burosumab had not been missed. Participants could continue to receive burosumab for up to an additional 96 weeks during the extension period. Key inclusion criteria were age 1-4 years at the time of informed consent; fasting serum phosphorus concentration of less than 0·97 mmol/L (3·0 mg/dL); serum creatinine 8·8-35·4 μmol/L (0·1-0·4 mg/dL); radiographic evidence of rickets (at least five participants were required to have a Thacher Rickets Severity Score of ≥1·5 at the knee); and a confirmed PHEX mutation or a variant of unknown significance in the patient or direct relative also affected with X-linked hypophosphataemia. Conventional therapy was stopped upon enrolment. The coprimary endpoints were safety and change from baseline to week 40 in fasting serum phosphorus concentrations. Changes in rickets severity from baseline to weeks 40 and 64 (assessed radiographically using Thacher Rickets Severity Score and an adaptation of the Radiographic Global Impression of Change), and recumbent length or standing height, were key secondary outcomes. This trial is registered with ClinicalTrials.gov, number NCT02750618, and is ongoing.Between May 16, 2016, and June 10, 2016, we enrolled 13 children with X-linked hypophosphataemia. All 13 children completed 64 weeks of treatment and were included in the efficacy and safety analysis; none exceeded 70 weeks of treatment at the time of analysis. Serum phosphorus least squares mean increase from baseline to week 40 of treatment was 0·31 mmol/L (SE 0·04; 95% CI 0·24-0·39; 0·96 mg/dL [SE 0·12]; p0·0001). All patients had at least one adverse event. 14 treatment-related adverse events, mostly injection site reactions, occurred in five children. One serious adverse event considered unrelated to treatment (tooth abscess) occurred in a child with a history of tooth abscess. All other adverse events were mild to moderate, except a severe food allergy considered unrelated to treatment. No instances of nephrocalcinosis or noteworthy changes in the results of a standard safety chemistry panel emerged. Total Thacher Rickets Severity Score decreased by a least squares mean of -1·7 (SE 0·1; p0·0001) from baseline to week 40 and by -2·0 (SE 0·1; p0·0001) by week 64. The Radiographic Global Impression of Change score also indicated significant improvement, with a least squares mean score of +2·3 (SE 0·1) at week 40 and +2·2 (0·1) at week 64 (both p0·0001). Mean length or standing height Z score was maintained from baseline to week 64.Burosumab had a favourable safety profile, increased serum phosphorus, and improved rickets and prevented early declines in growth in children aged 1-4 years with X-linked hypophosphataemia. These findings could substantially alter the treatment of young children with X-linked hypophosphataemia.Ultragenyx Pharmaceutical and Kyowa Kirin International.

Background: NF-κB essential modulator (NEMO), encoded by IKBKG, is necessary for activation of the ubiquitous transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Animal studies suggest NEMO is required for NF-κB mediated bone homeostasis, but this has not been thoroughly studied in humans. IKBKG loss-of-function mutation causes incontinentia pigmenti (IP), a rare X-linked disease featuring linear hypopigmentation, alopecia, hypodontia, and immunodeficiency. Single case reports describe osteopetrosis (OPT) in boys carrying hypomorphic IKBKG mutations. Method: We studied the bone phenotype in women with IP with evaluation of radiographs of the spine and non-dominant arm and leg; lumbar spine and femoral neck aBMD using DXA; μ-CT and histomorphometry of trans-iliac crest biopsy specimens; bone turnover markers; and cellular phenotype in bone marrow skeletal (stromal) stem cells (BM-MSCs) in a cross-sectional, age-, sex-, and BMI-matched case-control study. X-chromosome inactivation was measured in blood leucocytes and BM-MSCs using a PCR method with methylation of HpaII sites. NF-κB activity was quantitated in BM-MSCs using a luciferase NF-κB reporter assay. Results: Seven Caucasian women with IP (age: 24–67 years and BMI: 20.0–35.2 kg/m 2 ) and IKBKG mutation (del exon 4–10 (n = 4); c.460C>T (n = 3)) were compared to matched controls. The IKBKG mutation carriers had extremely skewed X-inactivation (>90:10%) in blood, but not in BM-MSCs. NF-κB activity was lower in BM-MSCs from IKBKG mutation carriers (n = 5) compared to controls (3094 ± 679 vs. 5422 ± 1038/μg protein, p < 0.01). However, no differences were identified on skeletal radiographics, aBMD, μ-architecture of the iliac crest, or bone turnover markers. The IKBKG mutation carriers had a 1.7–fold greater extent of eroded surfaces relative to osteoid surfaces (p < 0.01), and a 2.0–fold greater proportion of arrested reversal surface relative to active reversal surface (p < 0.01). Conclusion: Unlike mutation-positive males, the IKBKG mutation-positive women did not manifest OPT.

X-linked hypophosphatemia is characterized by increased secretion of fibroblast growth factor 23 (FGF-23), which leads to hypophosphatemia and consequently rickets, osteomalacia, and skeletal deformities. We investigated burosumab, a monoclonal antibody that targets FGF-23, in patients with X-linked hypophosphatemia.In an open-label, phase 2 trial, we randomly assigned 52 children with X-linked hypophosphatemia, in a 1:1 ratio, to receive subcutaneous burosumab either every 2 weeks or every 4 weeks; the dose was adjusted to achieve a serum phosphorus level at the low end of the normal range. The primary end point was the change from baseline to weeks 40 and 64 in the Thacher rickets severity total score (ranging from 0 to 10, with higher scores indicating greater disease severity). In addition, the Radiographic Global Impression of Change was used to evaluate rachitic changes from baseline to week 40 and to week 64. Additional end points were changes in pharmacodynamic markers, linear growth, physical ability, and patient-reported outcomes and the incidence of adverse events.The mean Thacher rickets severity total score decreased from 1.9 at baseline to 0.8 at week 40 with every-2-week dosing and from 1.7 at baseline to 1.1 at week 40 with every-4-week dosing (P0.001 for both comparisons); these improvements persisted at week 64. The mean serum phosphorus level increased after the first dose in both groups, and more than half the patients in both groups had levels within the normal range (3.2 to 6.1 mg per deciliter [1.0 to 2.0 mmol per liter]) by week 6. Stable serum phosphorus levels were maintained through week 64 with every-2-week dosing. Renal tubular phosphate reabsorption increased from baseline in both groups, with an overall mean increase of 0.98 mg per deciliter (0.32 mmol per liter). The mean dose of burosumab at week 40 was 0.98 mg per kilogram of body weight with every-2-week dosing and 1.50 mg per kilogram with every-4-week dosing. Across both groups, the mean serum alkaline phosphatase level decreased from 459 U per liter at baseline to 369 U per liter at week 64. The mean standing-height z score increased in both groups, with greater improvement seen at all time points with every-2-week dosing (an increase from baseline of 0.19 at week 64) than with every-4-week dosing (an increase from baseline of 0.12 at week 64). Physical ability improved and pain decreased. Nearly all the adverse events were mild or moderate in severity.In children with X-linked hypophosphatemia, treatment with burosumab improved renal tubular phosphate reabsorption, serum phosphorus levels, linear growth, and physical function and reduced pain and the severity of rickets. (Funded by Ultragenyx Pharmaceutical and Kyowa Hakko Kirin; ClinicalTrials.gov number, NCT02163577 ; EudraCT number, 2014-000406-35 ).

Sclerosteosis (SOST) refers to two extremely rare yet similar skeletal dysplasias featuring a diffusely radiodense skeleton together with congenital syndactyly. SOST1 is transmitted as an autosomal recessive (AR) trait and to date caused by ten homozygous loss-of-function mutations within the gene SOST that encodes the inhibitor of Wnt-mediated bone formation, sclerostin. SOST2 is transmitted as an autosomal dominant (AD) or AR trait and to date caused by one heterozygous or two homozygous loss-of-function mutation(s), respectively, within the gene LRP4 that encodes the sclerostin interaction protein, low-density lipoprotein receptor-related protein 4 (LRP4). Herein, we investigated two teenagers and one middle-aged man with SOST in three families living in the state of Tamil Nadu in southern India. Next generation sequencing of their genomic DNA using our high bone density gene panel revealed SOST1 in the teenagers caused by a unique homozygous nonsense SOST mutation (c.129C > G, p.Tyr43X) and SOST2 in the man caused by homozygosity for one of the two known homozygous missense LRP4 mutations (c.3508C > T, p.Arg1170Trp). He becomes the fourth individual and the first non-European recognized with SOST2. His clinical course was milder than the life-threatening SOST1 demonstrated by the teenagers who suffered blindness, deafness, and raised intracranial pressure, yet his congenital syndactyly was more striking by featuring bony fusion of digits. All three patients were from consanguineous families and heterozygosity for the SOST mutation was documented in the mothers of both teenagers. Thus, on the endogamous genetic background of Indian Tamils, SOST1 from sclerostin deficiency compared to SOST2 from LRP4 deactivation is a more severe and life-threatening disorder featuring complications due to osteosclerosis of especially the skull. In contrast, the syndactyly of SOST2 is particularly striking by involving bony fusion of some digits. Both the SOST and LRP4 mutations in this ethnic population likely reflect genetic founders.

We report auricular ossification (AO) affecting the elastic cartilage of the ear as a newly recognized feature of osteoprotegerin (OPG)-deficiency juvenile Paget disease (JPD). AO and auricular calcification refer interchangeably to rigid pinnae, sparing the ear lobe, from various etiologies. JPD is a rare Mendelian disorder characterized by elevated serum alkaline phosphatase activity accompanied by skeletal pain and deformity from rapid bone turnover. Autosomal recessive transmission of loss-of-function mutations within TNFRSF11B encoding OPG accounts for most JPD (JPD1). JPD2 results from heterozygous constitutive activation of TNFRSF11A encoding RANK. Other causes of JPD remain unknown. In 2007, we reported a 60-year-old man with JPD1 who described hardening of his external ears at age 45 years, after 4 years of treatment with bisphosphonates (BPs). Subsequently, we noted rigid pinnae in a 17-year-old boy and 14-year-old girl, yet pliable pinnae in a 12-year-old boy, each with JPD1 and several years of BP treatment. Cranial imaging indicated cortical bone within the pinnae of both teenagers. Radiologic studies of our three JPD patients without mutations in TNFRSF11B showed normal auricles. Review of the JPD literature revealed possible AO in several reports. Two of our JPD1 patients had experienced difficult tracheal intubation, raising concern for mineralization of laryngeal elastic cartilage. Thus, AO is a newly recognized feature of JPD1, possibly exacerbated by BP treatment. Elastic cartilage at other sites in JPD1 might also ossify, and warrants investigation.

Heritable disorders that feature high bone mass (HBM) are rare. The etiology is typically a mutation(s) within a gene that regulates the differentiation and function of osteoblasts (OBs) or osteoclasts (OCs). Nevertheless, the molecular basis is unknown for approximately one-fifth of such entities. NF-κB signaling is a key regulator of bone remodeling and acts by enhancing OC survival while impairing OB maturation and function. The NF-κB transcription complex comprises five subunits. In mice, deletion of the p50 and p52 subunits together causes osteopetrosis (OPT). In humans, however, mutations within the genes that encode the NF-κB complex, including the Rela/p65 subunit, have not been reported. We describe a neonate who died suddenly and unexpectedly and was found at postmortem to have HBM documented radiographically and by skeletal histopathology. Serum was not available for study. Radiographic changes resembled malignant OPT, but histopathological investigation showed morphologically normal OCs and evidence of intact bone resorption excluding OPT. Furthermore, mutation analysis was negative for eight genes associated with OPT or HBM. Instead, accelerated bone formation appeared to account for the HBM. Subsequently, trio-based whole exome sequencing revealed a heterozygous de novo missense mutation (c.1534_1535delinsAG, p.Asp512Ser) in exon 11 of RELA encoding Rela/p65. The mutation was then verified using bidirectional Sanger sequencing. Lipopolysaccharide stimulation of patient fibroblasts elicited impaired NF-κB responses compared with healthy control fibroblasts. Five unrelated patients with unexplained HBM did not show a RELA defect. Ours is apparently the first report of a mutation within the NF-κB complex in humans. The missense change is associated with neonatal osteosclerosis from in utero increased OB function rather than failed OC action. These findings demonstrate the importance of the Rela/p65 subunit within the NF-κB pathway for human skeletal homeostasis and represent a new genetic cause of HBM. © 2015 American Society for Bone and Mineral Research.

Lenz-Majewski hyperostotic dwarfism (LMHD) is an ultra-rare Mendelian craniotubular dysostosis that causes skeletal dysmorphism and widely distributed osteosclerosis. Biochemical and histopathological characterization of the bone disease is incomplete and nonexistent, respectively. In 2014, a publication concerning five unrelated patients with LMHD disclosed that all carried one of three heterozygous missense mutations in PTDSS1 encoding phosphatidylserine synthase 1 (PSS1). PSS1 promotes the biosynthesis of phosphatidylserine (PTDS), which is a functional constituent of lipid bilayers. In vitro, these PTDSS1 mutations were gain-of-function and increased PTDS production. Notably, PTDS binds calcium within matrix vesicles to engender hydroxyapatite crystal formation, and may enhance mesenchymal stem cell differentiation leading to osteogenesis. We report an infant girl with LMHD and a novel heterozygous missense mutation (c.829T>C, p.Trp277Arg) within PTDSS1. Bone turnover markers suggested that her osteosclerosis resulted from accelerated formation with an unremarkable rate of resorption. Urinary amino acid quantitation revealed a greater than sixfold elevation of phosphoserine. Our findings affirm that PTDSS1 defects cause LMHD and support enhanced biosynthesis of PTDS in the pathogenesis of LMHD.

Many skeletal and mineral metabolic diseases have a genetic basis, which may be a germline single gene abnormality (i.e., a monogenic or Mendelian disorder), a somatic single gene defect (i.e., a postzygotic mosaic disorder), or involve several genetic variants (i.e., oligogenic or polygenic disorders). Genetic mutations causing Mendelian diseases usually have a large effect (i.e., penetrance), whereas oligogenic or polygenic disorders are associated with several genetic variations, each of which may have small effects with greater or smaller contributions from environmental factors (i.e., multifactorial disorders). Recognition of monogenic hereditary disorders is of clinical importance, as it may lead to relevant and timely investigations with correct treatment for the patient, and the patient’s relatives. The diagnosis of monogenic skeletal disease requires an awareness of the great diversity of symptoms and signs that may be associated with the disorder and, as ever, clinical skill is required. Thus, the clinician will need to pursue a careful and systematic approach with detailed history and physical examination and appropriate laboratory evaluations that should lead to judicious use of the range of diagnostic tools available. Finally, the clinician will require an appreciation of the increasing number and complexity of molecular genetic tests available to ensure their appropriate use and interpretation. These considerations are reviewed in this chapter.

Hypophosphatasia (HPP) is the remarkably informative inborn error of metabolism and metabolic bone disease that features low serum alkaline phosphatase (ALP) activity (hypophosphat asemia ) due to loss-of-function mutation(s) of TNSALP ( ALPL ), the gene that encodes the “tissue nonspecific” (bone/liver) isoenzyme of ALP (TNSALP). Lessons from HPP revealed that TNSALP is a cell-surface phosphohydrolase and showed how it functions. Several phosphocompounds that are TNSALP natural substrates accumulate extracellularly in HPP, including inorganic pyrophosphate (PP i ), an inhibitor of hydroxyapatite (HA) crystal growth, and pyridoxal 5′-phosphate (PLP), the major circulating form of vitamin B 6 . The superabundance of extracellular PP i blocks mineralization of the skeleton after matrix vesicles (MVs) rupture and release their nascent HA crystals. This causes rickets or osteomalacia despite normal or sometimes elevated circulating levels of calcium and phosphorus and adequate vitamin D. Failure of the cementumcovering tooth roots to mineralize leads to premature exfoliation of deciduous teeth. However, HPP spans the greatest range of severity of all skeletal diseases. This is largely explained by autosomal dominant (AD) and autosomal recessive (AR) inheritance from among several hundred, typically missense, mutations of TNSALP . Unless treated, perinatal HPP kills rapidly due to profound skeletal hypomineralization. Infantile HPP presents postnatally, but before age 6 months with rickets, failure-to-thrive, and sometimes hypercalcemia or vitamin B 6 -dependent seizures. Progressive chest deformity often causes lethal pulmonary problems. When severe, childhood HPP features symptomatic rickets, premature loss of deciduous teeth, and chronic muscle weakness. Adult HPP typically presents in middle age with osteomalacia and pseudofractures and sometimes arthropathies involving PP i deposition. Odonto-HPP, likely the most prevalent HPP, features tooth loss without skeletal disease. AR inheritance accounts for the most severely affected patients, whereas AD and rarely AR transmission explain the mild forms. In 2015, substantially effective bone-targeted TNSALP replacement therapy (asfotase alfa) received regulatory approval multinationally, typically for pediatric-onset HPP—the last rickets/osteomalacia to await a medical treatment. Now, for a variety of disorders, excessive ALP activity in diseased tissues is implicated in ectopic mineralization.

Revelation that the RANKL/OPG/RANK/NF-κB signaling pathway is a key modulator of osteoclast (OC) formation and action began in the 1990s with the discoveries of three new members of the tumor necrosis factor (TNF) superfamily (RANKL, OPG, and RANK). Soon after, genetically altered mice disclosed importance for these proteins in bone biology. Binding of RANKL to RANK promotes osteoclastogenesis and increased skeletal remodeling mediated through NF-κB signaling, whereas coupling of RANKL instead to its “decoy receptor” OPG inhibits this pathway. Subsequently, the significance of these proteins for humans was highlighted by the discoveries that mutation of the genes that encode RANKL ( TNFSF11 ), OPG ( TNFRSF11B ), and RANK ( TNFRSF11A ) explain several ultrarare but highly instructive Mendelian diseases that alter osteoclastogenesis and OC action. Those that activate the pathway (familial expansile osteolysis, early-onset Paget’s disease of bone, expansile skeletal hyperphosphatasia, panostotic expansile bone disease, and juvenile Paget’s disease) resemble classic Paget’s disease of bone. Those that deactivate the pathway cause “OC-poor” osteopetrosis due to impaired OC formation. In 2016, neonatal high bone mass became the newest heritable entity of this pathway and revealed the first mutation identified within the five-member NF-κB complex itself. This chapter reviews the clinical and laboratory features as well as the etiology and pathogenesis of these intriguing disorders.

Purpose: In patients with XLH, high circulating FGF23 induces renal phosphate (Pi) wasting, hypophosphatemia, rickets, and short stature. Current treatment with oral phosphate/active vitamin D is suboptimal in many patients. Methods: In a Phase 2 study, we randomized 52 children with XLH (ages 5-12 years, ≤Tanner 2) to receive KRN23 subcutaneously, either biweekly …

Heritable disorders of disturbed renal phosphate handling are the most common causes of rickets and osteomalacia in developed countries. However, the molecular mechanisms underlying the biochemical abnormalities and abnormal bone/cartilage mineralization in these diseases are complex, and our current models are incomplete, albeit they have grown remarkably over the past decade. Isolated renal phosphate wasting and consequent low serum phosphorus concentrations may result from a variety of genetic disorders that include: autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemic rickets (XLH), and autosomal recessive hypophosphatemic rickets (ARHR). Seminal findings have confirmed a central role for the endocrine molecule, fibroblast growth factor 23 (FGF23), in the pathogenesis of these diseases. Understanding the inter-relationships between the gene mutations underlying these disorders and regulation of FGF23 production and proteolysis has provided new and unanticipated therapeutic targets for management of this constellation of diseases. These advances have resulted from ground-breaking studies in humans and animal models that promise to fuel further successful studies.

Hypophosphatasia (HPP) is the heritable metabolic disease characterized by impaired skeletal mineralization due to low activity of the tissue-nonspecific isoenzyme of alkaline phosphatase. Although HPP during growth often manifests with distinctive radiographic skeletal features, no validated method was available to quantify them, including changes over time. We created the Radiographic Global Impression of Change (RGI-C) scale to assess changes in the skeletal burden of pediatric HPP. Site-specific pairs of radiographs of newborns, infants, and children with HPP from three clinical studies of asfotase alfa, an enzyme replacement therapy for HPP, were obtained at baseline and during treatment. Each pair was scored by three pediatric radiologists ("raters"), with nine raters across the three studies. Intrarater and interrater agreement was determined by weighted Kappa coefficients. Interrater reliability was assessed using intraclass correlation coefficients (ICCs) and by two-way random effects analysis of variance (ANOVA) and a mixed-model repeated measures ANOVA. Pearson correlation coefficients evaluated relationships of the RGI-C to the Rickets Severity Scale (RSS), Pediatric Outcomes Data Collection Instrument Global Function Parent Normative Score, Childhood Health Assessment Questionnaire Disability Index, 6-Minute Walk Test percent predicted, and Z-score for height in patients aged 6 to 12 years at baseline. Eighty-nine percent (8/9) of raters showed substantial or almost perfect intrarater agreement of sequential RGI-C scores (weighted Kappa coefficients, 0.72 to 0.93) and moderate or substantial interrater agreement (weighted Kappa coefficients, 0.53 to 0.71) in patients aged 0 to 12 years at baseline. Moderate-to-good interrater reliability was observed (ICC, 0.57 to 0.65). RGI-C scores were significantly (p ≤ 0.0065) correlated with the RSS and with measures of global function, disability, endurance, and growth in the patients aged 6 to 12 years at baseline. Thus, the RGI-C is valid and reliable for detecting clinically important changes in skeletal manifestations of severe HPP in newborns, infants, and children, including during asfotase alfa treatment. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Summary Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2 σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gαq/11 and Gαi/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling., Graphical Abstract, Highlights • Disease-causing AP2σ mutants impair Gαq/11 and Gαi/o signaling by CaSR, a class C GPCR • AP2σ mutants impair trafficking of the CaSR • The CaSR can signal by a sustained endosomal pathway • CaSR differentially uses Gαq/11 and Gαi/o for cell-surface and endosomal signaling, Gorvin et al. show that the class C GPCR calcium-sensing receptor (CaSR) mediates signaling from plasma membranes using Gαq/11 and Gαi/o and from endosomes by using only Gαq/11. Adaptor protein-2 σ subunit (AP2σ) mutations impair CaSR internalization, leading to reduced sustained endosomal signaling and hypercalcemia in humans.

Seven young patients with X-linked hypophosphatemia (XLH, having inactivating PHEX mutations) were discovered to accumulate osteopontin (OPN) at the sites of defective bone mineralization near osteocytes − the so-called hallmark periosteocytic (lacunar) “halos” of XLH. OPN was also localized in the pericanalicular matrix extending beyond the osteocyte lacunae, as well as in the hypomineralized matrix of tooth dentin. OPN, a potent inhibitor of mineralization normally degraded by PHEX , is a member of a family of acidic, phosphorylated, calcium-binding, extracellular matrix proteins known to regulate dental, skeletal, and pathologic mineralization. Associated with the increased amount of OPN (along with inhibitory OPN peptide fragments) in XLH bone matrix, we found an enlarged, hypomineralized, lacuno-canalicular network – a defective pattern of skeletal mineralization that decreases stiffness locally at: i ) the cell-matrix interface in the pericellular environment of the mechanosensing osteocyte, and ii ) the osteocyte's dendritic network of cell processes extending throughout the bone. Our findings of an excess of inhibitory OPN near osteocytes and their cell processes, and in dentin, spatially correlates with the defective mineralization observed at these sites in the skeleton and teeth of XLH patients. These changes likely contribute to the dento-osseous pathobiology of XLH, and participate in the aberrant bone adaptation and remodeling seen in XLH.

Heritable forms of hypophosphatemic rickets (HR) include X-linked dominant (XLH), autosomal recessive, and autosomal dominant HR (from deactivating mutations in PHEX, DMP1 or ENPP1, and activating mutations in FGF23, respectively). Over 30 years, we have cared for 284 children with HR. For those 72 deemed sporadic XLH, we preliminarily reported mutation analysis for 30 subjects. Eleven had PHEX mutations. However, the remaining 19 lacked readily identifiable defects in PHEX, DMP1, or FGF23. In 2008, a novel single-base change near the polyadenylation (pA) signal in the 3′-UTR of PHEX was identified in XLH by other investigators. This c.*231A > G mutation is 3-bp upstream of the putative pA signal (AATAAA) in PHEX. Accordingly, we investigated whether this 3′-UTR defect accounted for HR in any of these 19 sporadic XLH patients. PCR amplification and sequencing of their 3′-UTR region showed the c.*231A > G mutation in four unrelated boys. Then, among an additional 22 of our 72 “sporadic” XLH patients, one boy and one girl were found to have the 3′-UTR defect, totaling six patients. Among these 52 sporadic XLH patients with PHEX analysis, 36 were girls and 16 were boys; ie, a ∼2:1 gender ratio consistent with XLH. However, finding five boys and only one girl with this 3′-UTR mutation presented an unexplained gender bias (p = 0.02). Haplotyping for the five boys, all reportedly unrelated, showed a common core haplotype suggesting a founder. Five of their six mothers had been studied clinically and biochemically (three radiologically). Remarkably, the seemingly unaffected mothers of four of these boys carried the 3′-UTR mutation. These healthy women had normal height, straight limbs, lacked the radiographic presentation of XLH, and showed normal or slight decreases in fasting serum Pi levels and/or TmP/GFR. Hence, PHEX c.*231A > G can masquerade as sporadic or X-linked recessive HR. © 2014 American Society for Bone and Mineral Research.

Among the high bone mass disorders, the osteopetroses reflect osteoclast failure that prevents skeletal resorption and turnover, leading to reduced bone growth and modeling and characteristic histopathological and radiographic findings. We report an 11-year-old boy with a new syndrome that radiographically mimics osteopetrosis (OPT), but features rapid skeletal turnover. He presented at age 21 months with a parasellar, osteoclast-rich giant cell granuloma. Radiographs showed a dense skull, generalized osteosclerosis and cortical thickening, medullary cavity narrowing, and diminished modeling of tubular bones. His serum alkaline phosphatase was >5000 IU/L (normal