Pyridoxine challenge reflects pediatric hypophosphatasia severity and thereby examines tissue-nonspecific alkaline phosphatase's role in vitamin B6 metabolism.

Alkaline phosphatase (ALP) is detected in most human tissues. However, ALP activity is routinely assayed using high concentrations of artificial colorimetric substrates in phosphate-free laboratory buffers at lethal pH. Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the ALP isoenzyme expressed in bone, liver, kidney, and elsewhere and is therefore designated "tissue-nonspecific" ALP (TNSALP). Consequently, HPP harbors clues concerning the biological function of this phosphohydrolase that is anchored onto the surface of cells. The biochemical signature of HPP features low serum ALP activity (hypophosphatasemia) together with elevated plasma levels of three natural substrates of TNSALP: i) phosphoethanolamine (PEA), a component of the linkage apparatus that binds ALPs and other proteins to the plasma membrane surface; ii) inorganic pyrophosphate (PPi), an inhibitor of bone and tooth mineralization; and iii) pyridoxal 5'-phosphate (PLP), the principal circulating vitameric form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving several hundred ALPL mutations underlies the remarkably broad-ranging expressivity of HPP featuring tooth loss often with muscle weakness and rickets or osteomalacia. Thus, HPP associates the "bone" isoform of TNSALP with biomineralization, whereas the physiological role of the "liver", "kidney", and other isoforms of TNSALP remains uncertain. Herein, to examine HPP's broad-ranging severity and the function of TNSALP, we administered an oral challenge of pyridoxine (PN) hydrochloride to 116 children with HPP. We assayed both pre- and post-challenge serum ALP activity and plasma levels of PLP, the B6 degradation product pyridoxic acid (PA), and the B6 vitamer pyridoxal (PL) that can enter cells. Responses were validated by PN challenge of 14 healthy adults and 19 children with metabolic bone diseases other than HPP. HPP severity was assessed using our HPP clinical nosology and patient height Z-scores. PN challenge of all study groups did not alter serum ALP activity in our clinical laboratory. In HPP, both the post-challenge PLP level and the PLP increment correlated (Ps < 0.0001) with the clinical nosology and height Z-scores (Rs = +0.6009 and + 0.4886, and Rs = -0.4846 and - 0.5002, respectively). In contrast, the plasma levels and increments of PA and PL from the PN challenge became less pronounced with HPP severity. We discuss how our findings suggest extraskeletal TNSALP primarily conditioned the PN challenge responses, and explain why they caution against overzealous B6 supplementation of HPP.

Hypophosphatasia: Vitamin B6 status of affected children and adults.

Hypophosphatasia (HPP) is the heritable dento-osseous disease caused by loss-of-function mutation(s) of the gene ALPL that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP is a cell-surface homodimeric phosphomonoester phosphohydrolase expressed in healthy people especially in the skeleton, liver, kidneys, and developing teeth. In HPP, diminished TNSALP activity leads to extracellular accumulation of its natural substrates including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), the principal circulating form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving >450 usually missense defects scattered throughout ALPL largely explains the remarkably broad-ranging severity of this inborn-error-of-metabolism. In 1985 when we identified elevated plasma PLP as a biochemical hallmark of HPP, all 14 investigated affected children and adults had markedly increased PLP levels. However, pyridoxal (PL), the dephosphorylated form of PLP that enters cells to cofactor many enzymatic reactions, was not low but often inexplicably elevated. Levels of pyridoxic acid (PA), the B6 degradation product quantified to assess B6 sufficiency, were unremarkable. Canonical signs or symptoms of B6 deficiency or toxicity were absent. B6-dependent seizures in infants with life-threatening HPP were later explained by their profound deficiency of TNSALP activity blocking PLP dephosphorylation to PL and diminishing gamma-aminobutyric acid synthesis in the brain. Now, there is speculation that altered B6 metabolism causes further clinical complications in HPP. Herein, we assessed the plasma PL and PA levels accompanying previously reported elevated plasma PLP concentrations in 150 children and adolescents with HPP. Their mean (SD) plasma PL level was nearly double the mean for our healthy pediatric controls: 66.7 (59.0) nM versus 37.1 (22.2) nM (P < 0.0001), respectively. Their PA levels were broader than our pediatric control range, but their mean value was normal; 40.2 (25.1) nM versus 39.3 (9.9) nM (P = 0.7793), respectively. In contrast, adults with HPP often had plasma PL and PA levels suggestive of dietary B6 insufficiency. We discuss why the B6 levels of our pediatric patients with HPP would not cause B6 toxicity or deficiency, whereas in affected adults dietary B6 insufficiency can develop.

Hypophosphatasia: Biochemical hallmarks validate the expanded pediatric clinical nosology.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism due to loss-of-function mutation(s) of the ALPL (TNSALP) gene that encodes the tissue non-specific isoenzyme of alkaline phosphatase (TNSALP). TNSALP represents a family of cell-surface phosphohydrolases differing by post-translational modification that is expressed especially in the skeleton, liver, kidney, and developing teeth. Thus, the natural substrates of TNSALP accumulate extracellularly in HPP including inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), the principal circulating form of vitamin B6. The superabundance of extracellular PPi regularly causes tooth loss, and when sufficiently great can lead to rickets or osteomalacia. Sometimes diminished hydrolysis of PLP engenders vitamin B6-dependent seizures in profoundly affected babies. Autosomal dominant and autosomal recessive inheritance from among >340 ALPL mutations identified to date, typically missense and located throughout the gene, largely explains the remarkably wide-ranging severity of HPP, greatest of all skeletal diseases. In 2015, our demographic, clinical, and DXA findings acquired over 25 years from 173 children and adolescents with HPP validated and expanded the clinical nosology for pediatric patients to include according to increasing severity "odonto" HPP, "mild childhood" HPP, "severe childhood" HPP, "infantile" HPP, and "perinatal" HPP. Herein, we assessed this expanded nosology using biochemical hallmarks of HPP. We evaluated exclusively data from the 165 preteenage HPP patients in this cohort to exclude potential effects from physiological changes in TNSALP levels across puberty. All patients had subnormal serum total and bone-specific ALP and elevated plasma PLP, and nearly all had excessive urinary PPi excretion. Only the PLP levels were unchanged across puberty. Mean levels of all four biomarkers correlated with HPP severity ranked according to the HPP nosology, but the data overlapped among all four patient groups. Hence, these four biochemical hallmarks represent both a sensitive and reliable tool for diagnosing children with HPP. Furthermore, the hallmarks validate our expanded clinical nosology for pediatric HPP that, with limitations, is an improved framework for conceptualizing and working with this disorder's remarkably broad-ranging severity.

Hypophosphatasia: validation and expansion of the clinical nosology for children from 25 years experience with 173 pediatric patients.

Hypophosphatasia (HPP) is caused by loss-of-function mutation(s) within the gene TNSALP that encodes the "tissue-nonspecific" isoenzyme of alkaline phosphatase (TNSALP). In HPP, inorganic pyrophosphate, an inhibitor of mineralization and substrate for TNSALP, accumulates extracellularly often leading to rickets or osteomalacia and tooth loss, and sometimes to craniosynostosis and calcium crystal arthropathies. HPP's remarkably broad-ranging expressivity spans stillbirth from profound skeletal hypomineralization to adult-onset dental problems or arthropathies without bone disease, which is largely explained by autosomal recessive versus autosomal dominant transmission from among several hundred, usually missense, TNSALP mutations. For clinical purposes, this expressivity has been codified according to absence or presence of skeletal disease and then patient age at presentation and diagnosis. Pediatric patients are reported principally with "odonto", "childhood", "infantile", or "perinatal" HPP. However, this nosology has not been tested using a cohort of patients, and the ranges of the clinical and laboratory findings have not been defined and contrasted among these patient groups. To evaluate the extant nosology for HPP in children, we assessed our 25 years experience with 173 pediatric HPP patients. Data were exclusively from inpatient studies. The childhood form of HPP was further designated "mild" or "severe". Here, we focused on demographic, clinical, and dual-energy X-ray absorptiometry parameters compared to data from healthy American children. The 173-patient cohort comprised 64 individuals with odonto HPP, 38 with mild childhood HPP, 58 with severe childhood HPP, and 13 with infantile HPP. None was a survivor of perinatal HPP. TNSALP analysis revealed a mutation(s) in all 105 probands tested. Thirteen mutations were unique. Most patients represented autosomal dominant inheritance of HPP. Mutant allele dosage generally indicated the disorder's severity. Gender discordance was found for severe childhood HPP; 42 boys versus 16 girls (p=0.006), perhaps reflecting parental concern about stature and strength. Key disease parameters (e.g., height, weight, numbers of teeth lost prematurely, grip strength, spine and hip bone mineral density) were increasingly compromised as HPP was designated more severe. Although data overlapped successively between the four patient groups, body size (height and weight) differed significantly. Thus, our expanded nosology for HPP in children organizes the disorder's broad-ranging expressivity and should improve understanding of HPP presentation, natural history, complications, and prognosis.

"Atypical femoral fractures" during bisphosphonate exposure in adult hypophosphatasia.

We report a 55-year-old woman who suffered atypical subtrochanteric femoral fractures (ASFFs) after 4 years of exposure to alendronate and then zolendronate given for "osteoporosis." Before alendronate treatment, she had low bone mineral density. After several months of therapy, metatarsal stress fractures began. Bisphosphonate (BP) administration was stopped following the ASFFs, and the adult form of hypophosphatasia (HPP) was diagnosed from low serum alkaline phosphatase (ALP) activity, high endogenous levels of two natural substrates for the "tissue-nonspecific" isoenzyme of ALP (TNSALP), and a heterozygous mutation within the gene that encodes this enzyme. Experience with other HPP families showed that her mutation (Arg71His) with a second defective TNSALP allele can cause severe HPP in infancy, and when heterozygous can cause mild HPP featuring premature loss of deciduous teeth in children. Because the skeletal disease of HPP results from extracellular accumulation of the TNSALP substrate inorganic pyrophosphate (PPi) and its inhibitory effect on mineralization, perhaps HPP patients or carriers will have adverse effects from BPs. BPs are analogues of PPi and can suppress bone turnover but also deactivate TNSALP. Our report is the first of BP exposure preceding ASFFs in adult HPP. To explore a potential role for TNSALP deactivation in ASFFs, mutation analysis of TNSALP should be studied in a cohort of these patients. Meanwhile, clinicians must suspect HPP when clinical or laboratory clues include premature loss of primary dentition, pseudofractures or recurrent poorly healing metatarsal stress fractures, a family history suggestive of HPP, or low serum ALP activity. If HPP is documented, BP treatment might be avoided. To establish the diagnosis of HPP, assays for two natural substrates for TNSALP and TNSALP mutation analysis are available in commercial laboratories. With positive findings, radiological or bone biopsy evidence of acquired osteomalacia would indicate the adult form of this inborn-error-of-metabolism.

Hypophosphatasia: nonlethal disease despite skeletal presentation in utero (17 new cases and literature review).

Hypophosphatasia (HPP) is caused by deactivating mutation(s) within the gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Patients manifest rickets or osteomalacia and dental disease ranging from absence of skeletal mineralization in utero to only loss of adult dentition. Until recently, HPP skeletal disease in utero was thought to always predict a lethal outcome. However, several reports beginning in 1999 emphasized a benign prenatal form of HPP (BP-HPP) where skeletal disease detected in utero had a mild postnatal course. Here we describe prenatal and postnatal findings of 17 additional BP-HPP patients among our 178 pediatric HPP patients. Their findings are compared with those of their siblings with HPP, carrier parents, and others with identical TNSALP mutations. New information concerning 7 previously published BP-HPP patients accompanies a review of the HPP literature. Among our 17 BP-HPP patients, prenatal ultrasound showed normal chest or abdominal circumferences where recorded. Sometimes, poor skeletal mineralization, fetal crowding, and third-trimester improvement were observed. Postnatally, extremity bowing further improved (13 patients). BP-HPP severity postnatally spanned the "infantile" to "odonto" HPP phenotypes, resembling our patients who harbored identical TNSALP mutation(s). Eight had autosomal dominant (AD) and 9 had autosomal recessive (AR) BP-HPP. Fourteen of our 15 mothers were HPP carriers or affected. Of the 41 cumulative BP-HPP patients (24 literature cases meriting a BP-HPP diagnosis since 1996 plus our 17 patients), 63% had AR BP-HPP. Maternally transmitted HPP involved 11 of the 13 total AD BP-HPP probands (p = 0.01), supporting a maternal in utero effect on the baby. Fetal crowding, normal fetal mineralization and chest size, and TNSALP heterozygosity seem to identify BP-HPP. However, bowed fetal long bones with AR HPP, specific TNSALP mutations, or poor skeletal mineralization before the third trimester do not reliably diagnose HPP lethality.