Hypophosphatasia (HPP) is the inborn error of metabolism that features low serum alkaline phosphatase (ALP) activity caused by loss-of-function mutation(s) within the gene for the tissue nonspecific isoenzyme of ALP (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate (PPi), a TNSALP substrate and inhibitor of mineralization, leads frequently to premature tooth loss and often to rickets or osteomalacia. In affected adults, the excess PPi sometimes also causes calcium pyrophosphate dihydrate (CPPD) deposition, PPi arthropathy, or pseudogout, or seemingly paradoxical deposition of hydroxyapatite crystals in ligaments or around joints when the condition is called calcific periarthritis (CP). We report three middle-aged sisters with CP as the only clinical manifestation of HPP. Each presented during early adult life with recurrent episodes of pain principally around the shoulders, elbows, wrists, hips, or Achilles tendon. Otherwise, they were in good health, including no history of unusual dental disease, fractures, or pseudofractures. Calcific deposits were identified in symptomatic areas principally by ultrasonographic assessment but also confirmed radiographically. All three sisters had low serum levels of total and bone-specific ALP, hyperphosphatemia, and increased serum concentrations of the TNSALP substrate pyridoxal 5′-phosphate together characteristic of HPP. Mutation analysis revealed that each carried a single unique 18-bp duplication within TNSALP (c.188_205dup18, p.Gly63_Thr68dup) as did two of their healthy sons and their mother, who was without signs of CPPD deposition or CP but had knee osteoarthritis. We find that CP can be the only complication of HPP in adults. Thus, multiple juxta-articular deposits of hydroxyapatite causing CP may be a useful sign of HPP, especially when the CP is familial. © 2014 American Society for Bone and Mineral Research.
Hypophosphatasia (HPP) is the rare heritable dento-osseous disorder characterized biochemically by low serum alkaline phosphatase (ALP) activity (hypophosphatasemia) and caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Both autosomal dominant and autosomal recessive inheritance involving more than 260 TNSALP mutations largely explain the remarkably broad-ranging severity of this inborn error of metabolism. The clinical expression of HPP spans from lethal absence of skeletal mineralization at birth to dental problems without bone disease presenting during childhood or adult life.[1, 3, 4]
In HPP, inorganic pyrophosphate (PPi), a natural substrate of TNSALP and inhibitor of mineralization, accumulates extracellularly and leads to rickets or osteomalacia in all but the mildest (“odonto”) form of the disease. In pediatric patients, the most consistent clinical manifestation is premature (ie, age <5 years) exfoliation of deciduous (“baby”) teeth attributable to deficiency of mineralized cementum covering the tooth roots. The adult form of HPP typically presents in middle age with recurrent poorly healing metatarsal stress fractures and proximal femoral pseudofractures,[7, 8] but sometimes there is a remote history of rickets or premature tooth loss in childhood. In adults with HPP, the PPi excess can also lead to calcium pyrophosphate dihydrate (CPPD) crystal deposition in cartilage (chondrocalcinosis), pseudogout, and pyrophosphate arthropathy.[9, 10]
In the non-HPP general population, periarticular deposits of hydroxyapatite (HA) around the shoulders and elsewhere are often asymptomatic.[11, 12] However, there may be pain and functional impairment, and then the condition is called calcific periarthritis (CP).[11, 12] Sites of CP include the shoulder, greater trochanter of the hip, epicondyle of the elbow, wrist (particularly near the pisiform), knee, and other locations. Disorders associated with CP include renal failure, local intra-articular corticosteroid injections with calcifications along the injection tract, and scleroderma (particularly CREST syndrome with subcutaneous deposits).[11, 12] Over the past 25 years, a few instances of CP have been reported in adults with HPP.[13-16] Nevertheless, HPP is not on the “checklist” of conditions associated with CP.
We report three middle-aged sisters with symptomatic CP as their only clinical manifestation of HPP.
Materials and Methods
Three Spanish sisters, aged 43 to 52 years, each presented during young adult life with recurrent episodes of pain at the shoulders, elbows, wrists, hips, or Achilles tendon and elsewhere. Otherwise, they reported good health. None had received a bisphosphonate.
The proposita (sister #1) was referred at age 50 years for symptoms of CP (Fig. 1). Among the sisters, she suffered the most severe, multifocal, and frequent episodes of pain. Subsequently, we also followed sister #2 because of chronic pain at multiple periarticular locations. Sister #3 had acute attacks of pain between intervals of good health lasting months. They were prescribed analgesics, anti-inflammatory drugs, and physiotherapy with poor relief of pain. Two sisters (#1 and #3) had arthroscopy of one shoulder with temporary relief of symptoms, but no specimens were looked at for crystals, etc.
When routine laboratory investigation showed that each sister was hypophosphatasemic, HPP was suspected. However, they gave no history of premature loss of deciduous teeth, growth retardation, weakness, bone pain, or fractures (sister #2 had a doubtful posttraumatic rib fracture at age 7 years).
To investigate the possibility of HPP, all three sisters underwent further radiographic studies, central dual-energy X-ray absorptiometry (DXA) measurements (Lunar Prodigy, Madison, WI, USA), and quantitation of two of the three substrates for TNSALP that accumulate extracellularly in HPP: phosphoethanolamine (PEA) in urine and pyridoxal 5′-phosphate (PLP) in plasma using high-pressure liquid chromatography in our hospital in Barcelona, Spain. Bone ALP (BAP) in serum was quantitated by Elisa (IDS, Vitro) with intra- and interassay coefficients of variation of 2.9% and 5.3%, respectively. TNSALP mutation analysis was also performed (see Mutation Studies below).
Subsequently, the extended family, except for the sisters' asymptomatic siblings who had normal serum ALP levels, was investigated for HPP (Fig. 1). Additional laboratory tests included quantitation of serum total ALP activity, bone ALP activity, plasma PLP concentration, and urinary PEA levels of the three sisters' mother and six of their eight children, aged 11 to 26 years, all of whom were healthy, including no history of premature loss of teeth.
After informed written consent, leukocyte DNA from sisters #1 to 3 was extracted using the Gentra Puregene DNA extraction kit (Invitrogen, Carlsbad, CA, USA) from peripheral blood sent air-express to St. Louis, MO, USA. TNSALP mutation analysis was performed in our research laboratory (SM and MPW, Washington University School of Medicine, St. Louis, MO, USA) by sequencing all coding exons and adjacent mRNA splice sites of this gene using previously described PCR and sequencing primers and conditions. After identification of a heterozygous TNSALP mutation in exon 4 in sisters #1 to 3, this same exon was sequenced for their mother and six of their eight children at the Immunology Department at Hospital Clinic, Barcelona, Spain.
TNSALP sequencing revealed one mutation in sisters #1 to 3, an 18-bp in-frame duplication in exon 4 (c.188_205dup18, p.Gly63_Thr68dup) that would insert a hexapeptide into one copy of their TNSALP but otherwise not disrupt the protein coding sequence. No second TNSALP mutation was identified. This heterozygous TNSALP duplication was subsequently found in the mother, one son of sister #1, and one son of sister #3 (Fig. 1).
Anteroposterior radiographs of the right shoulder and pelvis of sister #1 (Fig. 2A, B) demonstrated calcium deposition adjacent to the right greater tuberosity (likely within the rotator cuff) and adjacent to the right greater trochanter and bilateral ischial tuberosities (likely within the gluteal tendon insertion and hamstring tendon origins, respectively). Ultrasonographic assessment (Esaote, MyLab 70XV) of the symptomatic areas was then emphasized compared with radiography and showed calcific deposits for all three sisters. Sonographic images over the right greater trochanter in sister #1 (Fig. 2C) confirmed the presence of calcification within the gluteal tendon insertion. Similar, though less dramatic, periarticular calcifications were seen in sisters #2 and #3.
DXA of the three sisters showed unremarkable T-scores for L1 to L4 spine, total hip, and femoral neck (Table 1).
|Observation||Sister #1||Sister #2||Sister #3a|
|Premature loss of teeth||No||No||No|
|Fractures or pseudofractures||No||No (doubtful posttraumatic rib fissure at age 7 years)||No|
|Age at first symptom (pain) (years)||30||17||30|
|Chronic periarticular pain||Yes||Yes||No|
|Attacks of periarticular pain||Very frequentb||Frequentc||frequentc|
|Location of pain||Shoulders, elbows, wrists, hips, Achilles tendons||Shoulders, elbows, wrists, hips, knees||Shoulders, wrists, thumb, hips|
|BMD T-score (Z-score #3)|
|Lumbar spine (L1 to L4)||−1.4 (−0.4)||0.0 (+1.3)||(+1.9)|
|Total hip||−2.1 (−1.6)||−0.6 (+0.1)||(+0.2)|
|Femoral neck||−1.3 (−0.6)||0.1 (+1.0)||(+0.3)|
The sisters' mother reportedly had “osteoarthritis” (OA) with a long history of joint pain and surgery at one shoulder. She had low bone mineral density (BMD) by DXA, but our review of her radiographs (shoulder in 2007, knees in 2010) showed no calcific deposits to indicate CP or CPPD deposition. OA was confirmed in her knees.
The erythrocyte sedimentation rates and serum C-reactive protein levels of sisters #1 to 3 were normal in repeated measurements, and tests for antinuclear antibodies were negative.
Table 2 summarizes for sisters #1 to 3 the biochemical findings pertinent to HPP. Each had low serum total and bone-specific ALP levels, hyperphosphatemia, and increased plasma PLP levels that are together characteristic of HPP. Their urinary PEA levels were unremarkable, as may occur in mild HPP. Thus, in this sibship, elevated plasma PLP was a better marker vis-à-vis urine PEA levels for HPP. None of the sisters was hypercalcemic, which can complicate severe forms of HPP. Serum creatinine levels and estimated glomerular filtration rates were normal. The sisters' mother and the two sons who carried the TNSALP mutation (Fig. 1) showed hypophosphatasemia and high plasma PLP levels.
|Parameter||Normal range||Sister #1||Sister #2||Sister #3|
|Alkaline phosphatase (U/L)||80–250||72||59||39|
|Bone ALP (ng/mL)||7.4–15.3||7.2||5.1||2.3|
|Pyridoxal 5′-phosphate (nM)||15–96||237||103||151|
|25-hydroxyvitamin D (ng/mL)||≥30||24||34||20|
The typical presentation for adult HPP[7-9] seems to be recurrent poorly healing metatarsal stress fractures followed by femoral pseudofractures that resemble the prodromal lesions for “atypical subtrochanteric femoral fractures” in some patients with osteoporosis given antiresorptive therapy. Often, poorly characterized dental disease is already present. When severe, adult HPP can be debilitating from additional fractures elsewhere and generalized skeletal pain.[1, 7-9] Although the prevalences are not known, chondrocalcinosis, pseudogout, pyrophosphate arthropathy, and CP can also occur in adults with HPP.[13-16] Here, we found that CP can be the only clinical manifestation of HPP.
The first report of CP in HPP may have been in 1988 when Caspi and colleagues described an Iranian-Jewish family with three adult siblings who had multifocal symptomatic periarthritis and low levels in serum of ALP activity derived from liver. Unique deposition of basic calcium phosphate (octacalcium phosphate) crystals was documented but not accompanied by radiographic chondrocalcinosis. Slightly low BMD of the radial diaphysis was observed using an unspecified technique. There were no unusual dental findings. They concluded that the absence of unusual tooth problems, normal serum levels of bone ALP, and unremarkable spine radiographs failed to support HPP. Hence, the relationship between the periarticular calcifications and the low serum level of liver ALP activity seemed unclear. (TNSALP mutation causing HPP was discovered the year of their publication but was not studied for their patients).
In 1989, Chuck and colleagues reported three apparently unrelated cases of HPP who resembled our patients (without osteopenia) and presented with CP shown to be from HA deposition. They reviewed how low tissue levels of ALP activity can sometimes stimulate HA deposition, whereas still lower ALP levels inhibit HA formation (see below). Furthermore, they clarified how, unlike for HA, CPPD crystal formation is mainly restricted to collagenous loco-motor tissues (fibrocartilage and hyaline cartilage, tendon, capsule) and occurs in the metabolic diseases of hyperparathyroidism, hypomagnesemia, hemochromatosis, and Wilson's disease. It was uncertain whether their patients with CP represented a discrete mild subset of HPP without osteopenia or an early phase of classic HPP.
In 1990, Lassere and Jones described a mother and her two adult daughters with recurrent CP and a family tendency to develop generalized OA. Only one daughter had low serum ALP activity, which they called “HPP.” However, no typical clinical features of HPP were reported, and TNSALP mutation analysis was not performed.
In 2012, Iida and colleagues reported amelioration of pain after surgical resection of multiple inflamed periarticular calcifications involving the epicondyles of the elbows and knees in a 32-year-old man with mild hypophosphatasemia and phosphoethanolaminuria but no history of dento-osseous disease and normal BMD. His pain had not responded to NSAIDS and had become refractory to triamcinolone diacetate injections. Chemical classification of the crystal deposits was unsuccessful. He carried a heterozygous missense TNSALP mutation Phe310Leu, which is a common Japanese HPP mutation. Whether CP is prevalent in the Japanese with this TNSALP mutation is not known.
The three sisters who we studied for CP gave no history of dental or skeletal symptoms of HPP. CP was their only clinical manifestation of HPP and began in early adult life with intermittent periarticular pain that recurred thereafter. BMD Z-scores were unremarkable. In time, the classic dento-osseous complications of adult HPP may emerge, but their 78-year-old mother carries the same TNSALP mutation and has not had remarkable dental disease or fractures, etc.
Studies of HPP have revealed >260 distinctive mutations in the TNSALP gene. Missense defects, some with dominant/negative effects, account for approximately 75% of these mutations, whereas small insertions or deletions contribute about 15% (∼50% in-frame mutations). From her medical history and our review of her radiographs, our patients' mother did not have CP. She instead had OA of her knees. Furthermore, the son of sister #1 and one son of sister #3 also carried the TNSALP mutations, were young adults, but did not have symptoms of CP. Thus, the unique in-frame duplication of six amino acids in TNSALP of these three sisters was apparently not the complete explanation for their symptomatic CP. Other genetic or nongenetic factors seemed to contribute to the pathogenesis of this complication of HPP.
Molecular modeling of wild-type TNSALP has identified several important functional domains (active site, active site valley, homodimeric interface, crown domain, and calcium binding site) for this homodimeric enzyme. Although our patients' unique six amino acid duplication of TNSALP (p.Gly63_Thr68dup) would not appear to directly impact any of these domains, it is near amino acids that influence the active site and homodimeric interface. The mutation would create a bulge in the TNSALP protein that could perturb critical domains and disrupt enzymatic activity. Nevertheless, in the report of Iida and colleagues, CP occurred from a different TNSALP mutation. Hence, additional TNSALP mutations might partly explain future observations of CP in HPP kindreds.
The precise pathogenesis of CP in HPP is unclear, and this focal complication might seem paradoxical for an inborn error of metabolism characterized by a generalized defect in mineralization of the dentition and skeleton. PPi binds strongly to HA and these crystals then grow and dissolve slowly (“crystal poison”), understandably causing tooth loss from failure of cementum to mineralize and rickets in affected children and osteomalacia in adults from failure of osteoid to mineralize. However, at lesser concentrations, PPi seems to enhance calcium and Pi precipitation to form amorphous calcium phosphate.[1, 5, 14] Hence, if PPi levels are increased to such concentrations in certain tissues, PPi could stimulate HA deposition. In fact, sisters #1 to 3 have low but not the significantly diminished serum ALP values typically associated with skeletal manifestations of HPP. Sister #1, who was the most symptomatic from CP, had the least diminished serum ALP level. Perhaps the mild hypophosphatasemia together with hyperphosphatemia common in HPP of all three sisters explained their periarticular deposition of HA. Because our patients had no skeletal symptoms and BMD by DXA was essentially unremarkable for their ages, we did not perform iliac crest biopsies to search for the osteomalacia of adult HPP.
The other crystal-related arthropathy of adult HPP,[1, 9] CPPD deposition disease, probably results from extracellular excesses of PPi together with the failure of TNSALP to dissolve CPPD crystals.[22-24] Thus, the CPPD deposition, PPi arthropathy, and pseudogout of HPP seem to involve a different pathogenesis compared with the CP. In fact, none of the family members in our study who carried the TNSALP mutation showed radiologic evidence of CPPD deposition. Instead, CP was the only form of ectopic calcification and clinical manifestation of their HPP.
Accordingly, it is important to recognize that CP can be the only clinical complication of HPP to: 1) elude incorrect diagnoses, including the fairly numerous causes of hypophosphatasemia; 2) anticipate any associated dental or skeletal manifestations; and 3) perhaps explain the signs and symptoms of other family members. HPP should be considered when ectopic HA is discovered, particularly in a periarticular distribution and family cluster. This might help to avoid potentially harmful drugs, such as bisphosphonates used to treat osteoporosis. Instead, “off-label” use of teriparatide has had some success for adult HPP, and bone-targeted TNSALP-replacement therapy is emerging as a potential treatment for HPP. However, their effects on ectopic mineral (CPPD and CP) in HPP are not known.
MPW receives consulting fees and research grant support from Alexion Pharmaceuticals, Cheshire, CT, USA. All other authors state that they have no conflicts of interest.
We are grateful to Ms Sharon McKenzie and Ms Vivienne McKenzie for preparing the manuscript, Ms Margaret Huskey for TNSALP sequencing, and Dr Silvia Ruiz-Gaspá for technical support.
This study was supported by Shriners Hospitals for Children, The Clark and Mildred Cox Inherited Metabolic Bone Disease Research Fund, The Frederick S. Upton Foundation, The Hypophosphatasia Research Fund, and The Barnes-Jewish Hospital Foundation.
Authors' roles: All authors helped write and approved the manuscript. NG, IM, and PP diagnosed the patients, conducted family studies, and contributed to laboratory, radiological, DXA and ultrasonographic data collection. JLD reviewed the radiographs to characterize the radiological findings. SM and EGR performed the mutation analysis. MPW coordinated the study and the manuscript preparation.