Persistent idiopathic hyperphosphatasemia from bone alkaline phosphatase in a healthy boy.

Alkaline phosphatase (ALP) in humans comprises a family of four cell-surface phosphomonoester phosphohydrolase isozymes. Three genes separately encode the "tissue-specific" ALPs whereas the fourth gene encodes ubiquitous homodimeric "tissue-nonspecific" ALP (TNSALP) richly expressed in bone, liver, kidney, and developing teeth. TNSALP monomers have five putative N-linked glycosylation sites where different post-translational modifications account for this isozyme's distinctive physicochemical properties in different organs. Three bone-derived TNSALP (BALP) isoforms (B/I, B1, and B2) are present in healthy serum, whereas a fourth BALP isoform (B1x) can circulate in chronic kidney disease. Herein, we report a healthy boy with persistent hyperphosphatasemia due to BALP levels two- to threefold higher than age-appropriate reference values. High-performance liquid chromatography, electrophoresis, heat inactivation, catalysis inhibition, and polyethylene glycol precipitation revealed increased serum B/I, B1, and B2 differing from patterns found in skeletal diseases. B/I was ~23-fold elevated. Absence of mental retardation and physical stigmata excluded Mabry syndrome, the ALP-anchoring disorder causing hyperphosphatasemia. Routine biochemical studies indicated intact mineral homeostasis. Serum N-terminal propeptide of type I procollagen (P1NP) level was normal, but C-terminal cross-linking telopeptide of type I collagen (CTX) level was elevated. However, radiological studies showed no evidence for a generalized skeletal disturbance. Circulating pyridoxal 5'-phosphate, a TNSALP natural substrate, was not low despite the laboratory hyperphosphatasemia, thereby suggesting BALP phosphohydrolase activity was not elevated endogenously. Mutation analysis of the ALPL gene encoding TNSALP revealed no defect. His non-consanguineous healthy parents had serum total ALP activity and BALP protein levels that were normal. Our patient's sporadic idiopathic hyperphosphatasemia could reflect altered post-translational modification together with increased expression and/or impaired degradation of BALP.

Juvenile Paget's Disease From Heterozygous Mutation of SP7 Encoding Osterix (Specificity Protein 7, Transcription Factor SP7).

Juvenile Paget's disease (JPD) became in 1974 the commonly used name for ultra-rare heritable occurrences of rapid bone remodeling throughout of the skeleton that present in infancy or early childhood as fractures and deformity hallmarked biochemically by marked elevation of serum alkaline phosphatase (ALP) activity (hyperphosphatasemia). Untreated, JPD can kill during childhood or young adult life. In 2002, we reported that homozygous deletion of the gene called tumor necrosis factor receptor superfamily, member 11B (TNFRSF11B) encoding osteoprotegerin (OPG) explained JPD in Navajos. Soon after, other bi-allelic loss-of-function TNFRSF11B defects were identified in JPD worldwide. OPG inhibits osteoclastogenesis and osteoclast activity by decoying receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) away from its receptor RANK. Then, in 2014, we reported JPD in a Bolivian girl caused by a heterozygous activating duplication within TNFRSF11A encoding RANK. Herein, we identify mutation of a third gene underlying JPD. An infant girl began atraumatic fracturing of her lower extremity long-bones. Skull deformity and mild hearing loss followed. Our single investigation of the patient, when she was 15 years-of-age, showed generalized osteosclerosis and hyperostosis. DXA revealed a Z-score of +5.1 at her lumbar spine and T-score of +3.3 at her non-dominant wrist. Biochemical studies were consistent with positive mineral balance and several markers of bone turnover were elevated and included striking hyperphosphatasemia. Iliac crest histopathology was consistent with rapid skeletal remodeling. Measles virus transcripts, common in classic Paget's disease of bone, were not detected in circulating mononuclear cells. Then, reportedly, she responded to several months of alendronate therapy with less skeletal pain and correction of hyperphosphatasemia but had been lost to our follow-up. After we detected no defect in TNFRSF11A or B, trio exome sequencing revealed a de novo heterozygous missense mutation (c.926C>G; p.S309W) within SP7 encoding the osteoblast transcription factor osterix (specificity protein 7, transcription factor SP7). Thus, mutation of SP7 represents a third genetic cause of JPD.

Bruck syndrome 2 variant lacking congenital contractures and involving a novel compound heterozygous PLOD2 mutation.

Bruck syndrome (BRKS) is the rare disorder that features congenital joint contractures often with pterygia and subsequent fractures, also known as osteogenesis imperfecta (OI) type XI (OMIM # 610968). Its two forms, BRKS1 (OMIM # 259450) and BRKS2 (OMIM # 609220), reflect autosomal recessive (AR) inheritance of FKBP10 and PLOD2 loss-of-function mutations, respectively. A 10-year-old girl was referred with blue sclera, osteopenia, poorly-healing fragility fractures, Wormian skull bones, cleft soft palate, congenital fusion of cervical vertebrae, progressive scoliosis, bell-shaped thorax, restrictive and reactive pulmonary disease, protrusio acetabuli, short stature, and additional dysmorphic features without joint contractures. Iliac crest biopsy after alendronate treatment that improved her bone density revealed low trabecular connectivity, abundant patchy osteoid, and active bone formation with widely-spaced tetracycline labels. Chromosome 22q11 deletion analysis for velocardiofacial syndrome, COL1A1 and COL1A2 sequencing for prevalent types of OI, and Sanger sequencing of LRP5, PPIB, FKBP10, and IFITM5 for rare pediatric osteoporoses were negative. Copy number microarray excluded a contiguous gene syndrome. Instead, exome sequencing revealed two missense variants in PLOD2 which encodes procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (lysyl hydroxylase 2, LH2); exon 8, c.797G>T, p.Gly266Val (paternal), and exon 12, c.1280A>G, p.Asn427Ser (maternal). In the Exome Aggregation Consortium (ExAC) database, low frequency (Gly266Val, 0.0000419) and absence (Asn427Ser) implicated both variants as mutations of PLOD2. The father, mother, and sister (who carried the exon 12 defect) were reportedly well with normal parental DXA findings. BRKS2, characterized by under-hydroxylation of type I collagen telopeptides compromising their crosslinking, has been reported in at least 16 probands/families. Most PLOD2 mutations involve exons 17-19 (of 20 total) encoding the C-terminal domain with LH activity. However, truncating defects (nonsense, frameshift, splice site mutations) are also found throughout PLOD2. In three reports, AR PLOD2 mutations are not associated with congenital contractures. Our patient's missense defects lie within the central domain of unknown function of PLOD2. In our patient, compound heterozygosity with PLOD2 mutations is associated with a clinical phenotype distinctive from classic BRKS2 indicating that when COL1A1 and COL1A2 mutation testing is negative for OI without congenital contractures or pterygia, atypical BRKS should be considered.

New explanation for autosomal dominant high bone mass: Mutation of low-density lipoprotein receptor-related protein 6.

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/ß-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 ß-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 ß-propeller of LRP6. In Family 1 LRP6 c.602C>T, p.A201V was homologous to LRP5 HBM mutation c.641C>T, p.A214V, and in Family 2 LRP6 c.553A>C, p.N185H was homologous to LRP5 HBM mutation c.593A>G, 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.

Unique Variant of NOD2 Pediatric Granulomatous Arthritis With Severe 1,25-Dihydroxyvitamin D-Mediated Hypercalcemia and Generalized Osteosclerosis.

Pediatric granulomatous arthritis (PGA) refers to two formerly separate entities: autosomal dominant Blau syndrome (BS) and its sporadic phenocopy early-onset sarcoidosis (EOS). In 2001 BS and in 2005 EOS became explained by heterozygous mutations within the gene that encodes nucleotide-binding oligomerization domain-containing protein 2 (NOD2), also called caspase recruitment domain-containing protein 15 (CARD15). NOD2 is a microbe sensor in leukocyte cytosol that activates and regulates inflammation. PGA is characterized by a triad of autoinflammatory problems (dermatitis, uveitis, and arthritis) in early childhood, which suggests the causal NOD2/CARD15 mutations are activating defects. Additional complications of PGA were recognized especially when NOD2 mutation analysis became generally available. However, in PGA, hypercalcemia is only briefly mentioned, and generalized osteosclerosis is not reported, although NOD2 regulates NF-κB signaling essential for osteoclastogenesis and osteoclast function. Herein, we report a 4-year-old girl with PGA uniquely complicated by severe 1,25(OH)2 D-mediated hypercalcemia, nephrocalcinosis, and compromised renal function together with radiological and histopathological features of osteopetrosis (OPT). The classic triad of PGA complications was absent, although joint pain and an antalgic gait accompanied wrist, knee, and ankle swelling and soft non-tender masses over her hands, knees, and feet. MRI revealed tenosynovitis in her hands and suprapatellar effusions. Synovial biopsy demonstrated reactive synovitis without granulomas. Spontaneous resolution of metaphyseal osteosclerosis occurred while biochemical markers indicated active bone turnover. Anti-inflammatory medications suppressed circulating 1,25(OH)2 D, corrected the hypercalcemia, and improved her renal function, joint pain and swelling, and gait. Mutation analysis excluded idiopathic infantile hypercalcemia, type 1, and known forms of OPT, and identified a heterozygous germline missense mutation in NOD2 common in PGA (c.1001G>A, p.Arg334Gln). Thus, radiological and histological findings of OPT and severe hypercalcemia from apparent extrarenal production of 1,25(OH)2 D can complicate NOD2-associated PGA. Although the skeletal findings seem inconsequential, treatment of the hypercalcemia is crucial to protect the kidneys. © 2018 American Society for Bone and Mineral Research.

Melorheostosis: Exome sequencing of an associated dermatosis implicates postzygotic mosaicism of mutated KRAS.

Melorheostosis (MEL) is the rare sporadic dysostosis characterized by monostotic or polyostotic osteosclerosis and hyperostosis often distributed in a sclerotomal pattern. The prevailing hypothesis for MEL invokes postzygotic mosaicism. Sometimes scleroderma-like skin changes, considered a representation of the pathogenetic process of MEL, overlie the bony changes, and sometimes MEL becomes malignant. Osteopoikilosis (OPK) is the autosomal dominant skeletal dysplasia that features symmetrically distributed punctate osteosclerosis due to heterozygous loss-of-function mutation within LEMD3. Rarely, radiographic findings of MEL occur in OPK. However, germline mutation of LEMD3 does not explain sporadic MEL. To explore if mosaicism underlies MEL, we studied a boy with polyostotic MEL and characteristic overlying scleroderma-like skin, a few bony lesions consistent with OPK, and a large epidermal nevus known to usually harbor a HRAS, FGFR3, or PIK3CA gene mutation. Exome sequencing was performed to ~100× average read depth for his two dermatoses, two areas of normal skin, and peripheral blood leukocytes. As expected for non-malignant tissues, the patient's mutation burden in his normal skin and leukocytes was low. He, his mother, and his maternal grandfather carried a heterozygous, germline, in-frame, 24-base-pair deletion in LEMD3. Radiographs of the patient and his mother revealed bony foci consistent with OPK, but she showed no MEL. For the patient, somatic variant analysis, using four algorithms to compare all 20 possible pairwise combinations of his five DNA samples, identified only one high-confidence mutation, heterozygous KRAS Q61H (NM_033360.3:c.183A>C, NP_203524.1:p.Gln61His), in both his dermatoses but absent in his normal skin and blood. Thus, sparing our patient biopsy of his MEL bone, we identified a heterozygous somatic KRAS mutation in his scleroderma-like dermatosis considered a surrogate for MEL. This implicates postzygotic mosaicism of mutated KRAS, perhaps facilitated by germline LEMD3 haploinsufficiency, causing his MEL.

Auricular ossification: A newly recognized feature of osteoprotegerin-deficiency juvenile Paget disease.

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.

Lenz-Majewski hyperostotic dwarfism with hyperphosphoserinuria from a novel mutation in PTDSS1 encoding phosphatidylserine synthase 1.

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.

Multicentric carpotarsal osteolysis syndrome is caused by only a few domain-specific mutations in MAFB, a negative regulator of RANKL-induced osteoclastogenesis.

Multicentric carpotarsal osteolysis syndrome (MCTO), an autosomal dominant disorder that often presents sporadically, features carpal-tarsal lysis frequently followed by nephropathy and renal failure. In 2012, mutations in the single-exon gene MAFB were reported in 13 probands with MCTO. MAFB is a negative regulator of RANKL-mediated osteoclastogenesis. We studied nine MCTO patients (seven sporadic patients and one affected mother and son) for MAFB mutation. We PCR-amplified and selectively sequenced the MAFB region that contains the transactivation domain in this 323 amino acid protein, where mutations were previously reported for MCTO. We found five different heterozygous missense defects among eight probands: c.176C > T, p.Pro59Leu; c.185C > T, p.Thr62Ile; c.206C > T, p.Ser69Leu (four had this defect); c.209C > T, p.Ser70Leu; and c.211C > T, p.Pro71Ser. All 5 mutations are within a 13 amino acid stretch of the transactivation domain. Four were identical to the previously reported mutations. Our unique mutation (c.185C > T, p.Thr62Ile) involved the same domain. DNA available from seven parents of the seven sporadic patients did not show their child's MAFB mutation. The affected mother and son had an identical defect. Hence, the mutations for 7/8 probands were suspected to have arisen spontaneously as there was no history of features of MCTO in either parent. Penetrance of MCTO seemed complete. Lack of nonsense or other truncating mutations suggested a dominant-negative pathogenesis. Our findings indicate that only a few transactivation domain-specific mutations within MAFB cause MCTO.