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CLINICAL CASE SEMINAR |
Center for Metabolic Bone Disease and Molecular Research (M.P.W., S.M.), Shriners Hospitals for Children, St. Louis, Missouri 63131; Division of Bone and Mineral Diseases (M.P.W., S.M.), Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110; and Cleveland Clinic Foundation (C.D.), Cleveland Clinic Lerner College of Medicine at Case Western University, Cleveland, Ohio 44195
Address all correspondence and requests for reprints to: Dr. Michael P. Whyte, Shriners Hospitals for Children, 2001 South Lindbergh Boulevard, St. Louis, Missouri 63131-3597. E-mail: mwhyte{at}shrinenet.org.
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Abstract |
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Patient and Methods: A middle-aged woman sustained a slowly healing MTSF and then two enlarging MTSFs and a spontaneous proximal femur fracture. Pain persisted at all fracture sites. HPP was diagnosed as a result of low ALP activity (10–24 IU/liter; normal, 40–150 IU/liter) and elevated inorganic phosphate and pyridoxal 5'-phosphate concentrations in serum. Teriparatide (TPTD) (recombinant human PTH 1–34), 20 µg, was injected sc daily in an attempt to enhance osteoblast synthesis of TNSALP.
Results: Six weeks later, all fracture pain improved, and it resolved after 4 months. Radiographs of the enlarging MTSFs showed repair after 2–4 months. The femur fracture partially mended after 2 months and then healed. Additionally, hypophosphatasemia and hyperphosphatemia corrected, and biochemical markers of bone remodeling increased as long as TPTD (given for 18 months) was continued. The patient carried a heterozygous TNSALP missense mutation, p.D378V, which is common in the United States.
Conclusion: This first HPP patient given TPTD demonstrated fracture repair accompanying correction of hypophosphatasemia and hyperphosphatemia and bone marker responses indicating enhanced skeletal remodeling. Increased TNSALP synthesis in bone together with lowered extracellular concentrations of inorganic phosphate (a competitive inhibitor of ALPs) seemed to improve her skeletal mineralization. Further evaluation of TPTD for HPP is warranted.
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Introduction |
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In health, TNSALP is synthesized ubiquitously (particularly in bone, liver, and kidney) and is especially abundant on the surface of matrix vesicles formed by chondrocytes and osteoblasts (3). In HPP, the biochemical role of TNSALP is revealed (2) by extracellular accumulation of several phosphocompound substrates, including pyridoxal 5'-phosphate (PLP) (4) and inorganic pyrophosphate (PPi) (5). PPi is an inhibitor of hydroxyapatite crystal nucleation and growth (6, 7), and PPi excesses in HPP account for the characteristic rickets or osteomalacia (1, 2).
Additionally, HPP is associated with high-normal or elevated circulating levels of inorganic phosphate (Pi) from enhanced renal reclamation of filtered Pi (1, 2), and perhaps extracellular excesses of Pi, a competitive inhibitor of ALPs (8), indirectly contribute to the defective skeletal mineralization (9).
Nevertheless, HPP presentation ranges remarkably from death in utero with profound skeletal hypomineralization to spontaneous fractures beginning late in adult life (1). Five clinical forms are generally recognized, primarily according to patient age at diagnosis (1). In decreasing order of severity, patients are said to experience perinatal, infantile, childhood, adult, or odontohypophosphatasia. Actually, this nosology demarcates a remarkable continuum of HPP expressivity that is largely, but not completely (10), explained by the two inheritance patterns (autosomal-dominant or -recessive) (1) and the considerable number and variety of TNSALP defects (11). Adult HPP typically manifests in middle-age as a result of osteomalacia with recurrent, slowly healing metatarsal stress fractures (MTSFs) followed by painful, debilitating, proximal femur fractures or pseudofractures (12, 13, 14, 15). Pharmacotherapy remains elusive for all forms of HPP (1, 15, 16).
We describe the clinical, radiographic, and biochemical responses of a middle-aged woman with adult HPP given the recombinant PTH fragment, teriparatide (TPTD; recombinant human PTH 1–34).
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Patient and Methods |
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This 56-yr-old Caucasian woman had lost several "baby" teeth prematurely, typical of pediatric HPP, and then experienced numerous dental cavities during childhood. Estrogen replacement followed menopause, at age 40 yr, until age 53 yr. At age 54 yr, a spontaneous, painful, right fifth MTSF healed slowly over 7 months. Spontaneous right fourth and left fifth MTSFs occurred 2 yr later. When referred to us (C.D.), the right fourth MTSF had widened with extension of the fracture line over 5 months (Fig. 1
, A and B). The left fifth MTSF fracture had also widened. Her podiatrist planned open reduction and internal fixation of the breaks. Two months after referral, right anterior thigh pain began as a result of a spontaneous proximal femur fracture (Fig. 2, left panel).
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TNSALP sequencing, molecular modeling
After informed written consent approved by the Human Studies Committee, Washington University School of Medicine (St. Louis, MO), genomic DNA was extracted from blood leukocytes. All coding exons (no. 2–12) and adjacent mRNA splice sites of TNSALP were analyzed for mutations using our published methods (17). Computer modeling of the patient’s missense mutation affecting TNSALP (see below) was performed using RasMol version 2.7.2.1 (18).
TPTD treatment
TPTD, an anabolic peptide for bone used to treat patients with osteoporosis at high risk of fracture, stimulates osteoblast precursor cells and increases BSAP in the circulation (19).
After discussing with the patient the rationale for a trial of TPTD, injections began using 20 µg of Forteo (Eli Lilly and Co., Indianapolis, IN) administered sc daily (April 2004).
Bone remodeling was monitored by measuring serum osteocalcin and urinary NTX levels using fasting, morning, second-void collections. Bone density was quantitated by dual-energy x-ray absorptiometry (Lunar Prodigy, Madison, WI). Sequential radiographs assessed fracture healing.
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Results |
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After 6 wk of TPTD injections (hereafter referred to as TPTD), the patient reported 90 and 50% reduction in foot and right thigh pain, respectively, and after 4 months she was pain free.
After 5 months, TPTD was withheld for 2 months when biochemical studies suggested unfavorable uncoupling of bone turnover (NTX increased from 35 to 94 nM bone collagen equivalents/millimole creatinine, whereas osteocalcin decreased from 8.2 to 5.3 ng/ml) (Table 1
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TPTD resumed when she reported fatigue, fracture site pain, and discomfort throughout her skeleton, and serum osteocalcin decreased further to 2.8 ng/ml (Table 1). With resumption of TPTD, symptoms again promptly resolved.
TPTD was stopped after 16 months based on radiographic healing of the fractures, continued absence of fracture site pain, and sustained increases in serum ALP and osteocalcin and urinary NTX levels (Table 1).
A rise in serum creatinine from 1.0 to 1.4 mg/dl 18 months after beginning TPTD was attributed to celecoxib (Celebrex; Pfizer, New York, NY), 200 mg twice a day, prescribed for osteoarthritis. After a change to meloxican (Mobic; Boehringer Ingelheim Pharmaceuticals, Ingelheim, Germany), 15 mg daily, the value returned to 1.0 mg/dl.
Now, 8 months off TPTD, she remains free of all fracture site pain, contrasting with recurrence of pain when TPTD was withheld after 5 months of injections.
Radiological studies
Radiographs of the right fourth MTSF that had widened and extended (Fig. 1, A and B) showed obvious bony bridging and reduction of the fracture line after 2 months of TPTD (Fig. 1C).
Radiographs of the left fifth MTSF, sustained 7 months before TPTD, showed widening 2 months before TPTD. After 2 months of TPTD, calcification appeared with further callus after 4 and 7 months, and by 10 months, approximately 50% of the fracture line was gone with significant periosteal bridging. Radiographs at her last visit showed almost complete healing.
Radiographs of the femur fracture showed significant improvement between 2 and 4 months of TPTD (Fig. 2).
Dual-energy x-ray absorptiometry, 7 months before TPTD, revealed anteroposterior lumbar spine bone mineral density (BMD) of 1.470 g/cm2 with an elevated Z-score +2.9 and T-score +2.4, consistent with osteomalacia (20). Two years later, BMD was 4.3% lower with Z-score +2.5 and T-score +1.9.
Baseline left total hip BMD was 1.142 g/cm2 with Z-score +1.5 and T-score +1.1. Two years later, BMD was 4.1% higher with Z-score +2.0 and T-score +1.4.
Right total hip BMD (before fracture) was 1.108 g/cm2 with Z-score +1.2 and T-score +0.8 and showed essentially no change despite a nearby fracture and TPTD.
Biochemical findings
Biochemical testing indicated enhanced bone remodeling during TPTD (Table 1). Serum ALP increased from 24 IU/liter to a low-normal value of 46 IU/liter, fell to 27 IU/liter 3 months after TPTD was stopped, and then to 22 IU/liter 8 months later. Both osteocalcin and NTX increased overall during TPTD therapy.
During TPTD, hyperphosphatemia corrected from a maximum serum Pi level of 5.2 mg/dl to a nadir of 3.8 mg/dl, and remained normal at 4.0 mg/dl 3 months after stopping TPTD, but was once again elevated at 4.8 mg/dl 8 months later. Serum PLP levels seemed unaffected by TPTD (Table 1).
Before TPTD, serum calcium was 10.5 mg/dl (normal, 8.5–10.5 mg/dl), PTH was 31 pg/dl (normal, 10–60 pg/dl), and 1,25-dihydroxyvitamin D (Specialty Labs) was 42 pg/ml (normal, 25–66 pg/ml) while taking 1200-mg calcium supplementation with vitamin D2 daily accompanying 1200 mg calcium each day in her diet. Mild hypercalcemia (10.8 mg/dl) occurred once during TPTD with five subsequent normal values 10.2 to 10.5 mg/dl while then receiving 600-mg calcium supplementation daily. At her last visit taking TPTD, serum calcium was 10.6 mg/dl and 10.1 mg/dl 3 months later. Urine calcium, in a 24-h collection, was 352 mg (normal, 50–300 mg) just before TPTD was stopped and 175 mg/d off TPTD and calcium supplementation. Serum 1,25-dihydroxyvitamin D was 71 pg/ml after 2 months of TPTD but 31 pg/ml 5 and 8 months later.
TNSALP mutation analysis
Our patient proved heterozygous for a single TNSALP missense mutation (c.1133A>T, p.D378V) in exon 10 that we often identify in patients with HPP from throughout the United States (21). This nomenclature is from Den Dunnen and Antonarakis (22) with p.D378V, formerly called D361V.
Molecular modeling of wild-type TNSALP(D378) predicted that our patient’s missense amino acid substitution occurs at a Zn2+ coordination site near the active site of the isoenzyme (Fig. 3) (23).
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Discussion |
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TopAbstractIntroductionPatient and MethodsResultsDiscussionReferences |
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Our patient with adult HPP provided both subjective and objective evidence of benefit from TPTD, whereas her transient hypercalcemia and hypercalciuria seemed partly related to calcium supplementation, and her brief increase in serum creatinine seemed related to treatment with celecoxib. First, reduction of chronic pain at MTSFs was reported after 6 wk of therapy with prompt resolution of all skeletal discomfort as long as TPTD continued. Second, radiographs documented mending of MTSFs where extension and widening had previously occurred. Instead of forming a pseudofracture, the femur break went on to heal. Hence, a "placebo effect," common for skeletal pain, was unlikely. Similarly, spontaneous healing of the MTSFs seemed improbable. Clinical and radiographic features of infantile HPP can inexplicably improve (discussed in Refs. 1 and 16), but adult HPP features lingering difficulties, including proximal femoral pseudofractures (12, 13, 14, 15). Third, biochemical studies documented increases in serum ALP activity to the low-normal range, and levels of other markers of bone turnover were enhanced, suggesting accelerated skeletal remodeling. Fourth, when TPTD was stopped, pain did not recur at the fracture sites, but there was mild generalized bone discomfort 8 months later that did not require resumption of injections. Finally, biochemical findings indicated gradual loss of TNSALP effect with a steady fall in serum ALP and urine NTX levels and a return of hyperphosphatemia after therapy was stopped.
During TPTD, lumbar spine BMD decreased, whereas total hip BMD in the unfractured femur increased. Why this was observed is uncertain, but perhaps it reflected healing osteomalacia. However, this remains speculation because we did not use iliac crest histomorphometry after tetracycline labeling to document improved bone mineralization.
Genetic basis of HPP
To date, 178 different loss-of-function mutations in TNSALP (79% missense) have been identified exclusively in HPP (11). Severe forms of HPP are generally inherited as an autosomal-recessive trait, whereas milder forms can be transmitted as either an autosomal-dominant or autosomal-recessive trait (1, 10, 15, 16, 21).
We often encounter our patient’s heterozygous TNSALP missense mutation (c.1133A>T, p.D378V) in patients with HPP from throughout the United States and find that it typically causes a mild autosomal-dominant HPP phenotype, including adult HPP (21). Others have identified D378V in a family, apparently German, with autosomal-dominant early tooth loss (24). However, in one of our patients, an American girl with infantile HPP who lived 8 months, p.D378V occurred together with c.571G>A, p.E191K (25).
Mechanism of TPTD effect in adult HPP
Based on the crystal structure of placental ALP, a molecular model has been created for functional (dimeric) TNSALP (18, 26, 27). Several specific, homologous TNSALP domains are predicted, including an active site, active site valley, homodimer interface, crown domain, and metal binding sites (18, 27). Wild-type TNSALP amino acid residue D378 would be in the active site vicinity and function in Zn2+ coordination (18, 27).
Others have reported, using in vitro transfection, that our patient’s p.D378V missense mutation is translated and transported normally within cells, but retains less than 1% wild-type activity (18) and can inhibit the wild-type monomer (dominant negative effect), predicting an autosomal-dominant HPP phenotype (24). Our patient’s improvement during TPTD has two plausible mechanisms.
First, her increased serum ALP activity measured in the clinical laboratory seemed to reflect enhanced skeletal biosynthesis of TNSALP. Although we did not follow BSAP levels, other biochemical markers of bone turnover were also higher. The relatively strong inverse correlation between the expressivity of HPP and serum total ALP or BSAP activity (1, 28) suggests that even small increments of enzyme activity in bone would be beneficial. Furthermore, transfection studies of various TNSALP mutations indicate that slight increases in TNSALP activity in the skeleton could rescue severely affected patients with HPP (28). These observations are supported by marrow cell transplantation studies for HPP in which significant clinical and radiographic improvement occurred without alterations in circulating ALP or PLP levels (16, 29). Our patient’s serum PLP level seemed unchanged during TPTD, in keeping with a TPTD effect directly on the skeleton, because deficient TNSALP activity in the liver continued to account for her elevated circulating PLP levels (1, 2, 4). In theory, TPTD could have also induced a greater proportion of wild-type, homodimeric TNSALP in our patient’s osteoblasts by enhanced expression and/or cellular processing of the normal allele (Fig. 3).
Second, hyperphosphatemia characteristic of HPP (1, 2) corrected in our patient during TPTD, perhaps from its phosphaturic effect and/or from enhanced skeletal uptake of Pi during healing of her presumed osteomalacia. Adults with HPP have serum Pi levels above age-matched reference means, and not infrequently Pi values are elevated (1, 2). Although we did not study our patient’s renal Pi handling, enhanced renal reclamation of Pi (increased TmP/GFR) is the likely explanation (1, 2). In severe HPP, circulating PTH levels can be low and Pi concentrations high as a result of hypercalcemia (1, 2). Of interest, hypophosphatemia can occur in generalized arterial calcification of infancy in which extracellular PPi levels are low (30), suggesting that perhaps endogenous PPi levels condition renal reclamation of Pi. In any event, because Pi competitively inhibits ALPs (8), increased extracellular Pi levels in HPP could further compromise TNSALP activity (9). Perhaps, lowering circulating (extracellular) Pi concentrations (vis-a-vis TPTD) enhanced endogenous TNSALP activity in our patient (9).
TPTD for other patients with HPP?
Although all TNSALP mutations probably reduce TNSALP catalytic activity (3, 11), some also prevent the enzyme from reaching the cell surface (31, 32, 33) or perhaps decrease the stability of the monomer or dimer. Accordingly, TPTD responsiveness may differ considerably among patients with HPP. TPTD may prove most beneficial for dominantly inherited, mild forms of HPP, like in our patient, because wild-type TNSALP expression could be up-regulated. TPTD might be appropriate for patients with HPP with autosomal-recessive disease only if their TNSALP mutations do not abrogate enzymatic activity, prevent localization of the TNSALP dimer to matrix vesicles, or destroy its stability there. In 1986, we reported remarkable, transient correction of severe infantile HPP after empiric therapy, which included brief use of bovine PTH 1–34 (34), in which our molecular studies recently revealed that the patient was homozygous for a TNSALP missense mutation c.1348C>T, p.R433C (10). However, for compound heterozygous TNSALP defects, the complex interactions of both mutated alleles may preclude any TPTD responsiveness, requiring empiric assessments. For severe autosomal-recessive HPP (perinatal or infantile form), a positive response to TPTD is even less likely when essentially null mutations combine with no hope of promoting biosynthesis of functional TNSALP or enhancing catalytic activity by reductions in extracellular Pi concentrations. For such patients, TNSALP replacement targeted to bone or marrow cell transplantation may be required (16, 29, 35).
TPTD is currently contraindicated for pediatric patients because of concern for osteosarcoma developing within growth plates (19). So, until more is known, TPTD would not be given to children with mild HPP. However, investigation of TPTD might be acceptable for pediatric patients with debilitating HPP.
Conclusions
Further careful evaluation and reporting of TPTD for HPP, complemented by TNSALP mutation analysis, will be needed to better assess recombinant PTH treatment for this inborn error of metabolism. Hopefully, radiographic evaluation will document healing of rickets in affected children and that pseudofractures mend in affected adults. Iliac crest biopsy specimens, obtained after tetracycline labeling, will be important to demonstrate and quantify healing of osteomalacia in adult patients with HPP. Randomized, crossover studies might be most informative for future trials of TPTD for HPP.
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Acknowledgments |
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Footnotes |
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Results of this work were presented in part at the 27th Annual Meeting of The American Society for Bone and Mineral Research, September 23–27, 2005, Nashville, Tennessee [J Bone Miner Res 20 (Suppl 1):S100, 2005] and the 55th Annual Meeting of The American Society of Human Genetics, October 25–29, 2005, Salt Lake City, Utah [Proceedings, p 57, 2005].
Disclosure Statement: The authors have nothing to declare.
First Published Online January 9, 2007
Abbreviations: ALP, Alkaline phosphatase; BMD, bone mineral density; BSAP, bone-specific ALP; HPP, hypophosphatasia; MTSF, metatarsal stress fracture; NTX, N-telopeptide of type I collagen; Pi, inorganic phosphate; PLP, pyridoxal 5'-phosphate; PPi, inorganic pyrophosphate; TNSALP, tissue-nonspecific ALP; TPTD, teriparatide.
Received August 29, 2006.
Accepted January 2, 2007.
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References |
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TopAbstractIntroductionPatient and MethodsResultsDiscussionReferences |
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