Tissue-nonspecific Alkaline Phosphatase Gene Research Articles

Tissue-non-specific alkaline phosphatase mRNA expression and alkaline phosphatase activity following application of retinoic acid in cultured human dental pulp cells

Retinoic acid is a potent inducer of tissue-non-specific alkaline phosphatase (TNSALP) expression in various osteoblastic and fibroblastic cells, and may be involved in morphogenesis, cellular growth and differentiation. This study investigates the effects of retinoic acid on alkaline phosphatase activity and TNSALP gene expression in human dental pulp cells. Cultured cells were treated with various concentrations of retinoic acid (0, 10 −7, 10 −6, 10 −5 M) in 0.5% bovine serum albumin without serum. Alkaline phosphatase activity was determined by the rate of p-nitrophenyl phosphate hydrolysis and was also assayed in the presence of various inhibitors and under thermal inactivation. A set of specific oligonucleotide primers was selected, based on the nucleotide sequences of two human TNSALP mRNA (bone and liver) types, and reverse transcription-polymerase chain reaction (RT-PCR) performed. Inhibitory and thermal inactivation experiments revealed that the elevated alkaline phosphatase activity had properties of the TNSALP type. RT-PCR showed that retinoic acid enhanced the expression of bone-type TNSALP mRNA in pulp cells. However, the liver-type TNSALP mRNA was not detected. These findings suggest that the high alkaline phosphatase activity of retinoic acid-treated dental pulp cells is associated with increased transcription of the bone-type mRNA of the TNSALP gene and not with liver-type.

GABA and epileptogenesis: comparing gabrb3 gene-deficient mice with Angelman syndrome in man

The GABAergic system has long been implicated in epilepsy with defects in GABA neurotransmission being linked to epilepsy in both experimental animal models and human syndromes ( Olsen and Avoli, 1997). However, to date no human epileptic syndrome has been directly attributed to an altered GABAergic system. The observed defects in GABA neurotransmission in human epileptic syndromes may be the indirect result of a brain besieged by seizures. The use of animal models of epilepsy has sought to address these matters. The advent of gene targeting methodologies in mice now allows for a more direct assessment of GABA’s involvement in epileptogenesis. To date several genes associated with the GABAergic system have been disrupted. These include the genes for glutamic acid decarboxylase, both the 65- and 67-kDa isoforms (GAD65 and GAD67), the tissue non-specific alkaline phosphatase gene (TNAP) and genes for the GABA A receptor subunits α 6, β 3, γ 2, and δ (gabra6, gabrb3, gabrg2, and gabrd respectively). Gene disruptions of either GAD67 or gabrg2 result in neonatal lethality, while others, GAD65, TNAP, and gabrb3 exhibit increased mortality and spontaneous seizures. GABA receptor expression has been found to be both regionally and developmentally regulated. Thus in addition to their obvious role in controlling excitability in adult brain, a deficit in GABAergic function during development could be expected to elicit pleiotropic neurodevelopmental abnormalities perhaps including epilepsy. The GABA A receptor β 3 subunit gene, gabrb3/GABRB3 (mouse/human), is of particular interest because of its expression early in development and its possible role in the neurodevelopmental disorder Angelman syndrome. Individuals with this syndrome exhibit severe mental retardation and epilepsy. Mice with the gabrb3 gene disrupted likewise exhibit electroencephalograph (EEG) abnormalities, seizures, and behavioral characteristics typically associated with Angelman syndrome. These gabrb3 gene knockout mice provide direct evidence that a reduction of a specific subunit of the GABA A receptor system can result in epilepsy and support a GABAergic role in the pathophysiology of Angelman syndrome.

Correlation of alkaline phosphatase (ALP) determination and analysis of the tissue non-specific ALP gene in prenatal diagnosis of severe hypophosphatasia

Prenatal diagnosis of severe hypophosphatasia by mutation analysis of the tissue non-specific alkaline phosphatase (TNSALP) gene is reliable and mostly informative. However, alkaline phosphatase (ALP) assay of CVS may be a useful complementary and independent method, especially when a mutation is unidentified and DNA from the index case is unavailable, rendering impossible the use of DNA polymorphisms as genetic markers of the disease. We report here mutation analysis of the TNSALP gene and ALP assay in nine cases of prenatal diagnosis of severe hypophosphatasia. The results showed a good correlation between ALP assay and DNA analysis in all but one case, which suggested that in at least some cases low values of ALP may correspond to affected fetuses as well as to heterozygotes.

Correlation of alkaline phosphatase (ALP) determination and analysis of the tissue non-specific ALP gene in prenatal diagnosis of severe hypophosphatasia.

Prenatal diagnosis of severe hypophosphatasia by mutation analysis of the tissue non-specific alkaline phosphatase (TNSALP) gene is reliable and mostly informative. However, alkaline phosphatase (ALP) assay of CVS may be a useful complementary and independent method, especially when a mutation is unidentified and DNA from the index case is unavailable, rendering impossible the use of DNA polymorphisms as genetic markers of the disease. We report here mutation analysis of the TNSALP gene and ALP assay in nine cases of prenatal diagnosis of severe hypophosphatasia. The results showed a good correlation between ALP assay and DNA analysis in all but one case, which suggested that in at least some cases low values of ALP may correspond to affected fetuses as well as to heterozygotes.

Hypophosphatasia: diagnostic application of linked DNA markers in the dominantly inherited adult form

Hypophosphatasia is a rare disease characterized by low serum levels of tissue non-specific alkaline phosphatase (TNSALP) and a spectrum of skeletal disease varying from the severest form with death in utero to mild with no clinical abnormality in adults. Currently, the diagnosis of hypophosphatasia is made on the basis of clinical findings, radiography, low serum alkaline phosphatase levels and raised abnormal phosphorylated metabolites; there are elevations in serum pyridoxal 5'-phosphate, urinary phosphoethanolamine and inorganic pyrophosphate. In borderline cases the biochemical diagnosis remains uncertain. Prenatally, diagnosis is made using radiography and ultrasonography together with chorionic villus tissue biopsy, in which TNSALP levels are measured using an antibody-based assay. Since hypophosphatasia results from mutations in the TNSALP gene we have, for the first time in two U.K. families, undertaken restriction fragment length polymorphism (RFLP) analysis using three intragenic RFLPs for BclI and MspI at the ALPL locus. One family was informative, and a mutant-allele-specific haplotype with respect to three RFLPs was defined. In the other family the disease was shown to segregate with one allele of the BclI RFLP, but the MspI RFLPs were not informative. The disease segregated in the two families with different alleles of the BclI RFLP, suggesting that the mutations are likely to be different. We confirm that DNA analysis is likely to be the way ahead for diagnosing hypophosphatasia, and that standardized screening methods need to be developed for detecting mutations in these and other families.

Root development in mice lacking functional tissue non-specific alkaline phosphatase gene: inhibition of acellular cementum formation.

Tissue non-specific alkaline phosphatase (TNAP) is richly present in developing teeth including the cells of the periodontal ligament. Here, we investigated tooth and root development in mice lacking the TNAP gene. Heterozygous mutants were obtained from The Jackson Laboratory, Animal Resources (Bar Harbor, ME, USA) and bred. TNAP-deficient mice and their littermates were killed from 6 to 25 days after birth and their molar blocks processed for light and electron microscopy. It was observed that the eruption of the incisors into the oral cavity was delayed for 2 to 3 days. Also, the onset of mineralization of the mantle dentin in the roots of the developing molars was delayed for 2 to 3 days. Yet, dentin and enamel formation in the homozygous mutants showed a more or less normal pattern, with the exception of localized enamel hypoplasias. The most conspicuous finding was the defective formation of acellular cementum along the molar roots. Instead of a continuous layer, the cementum was deposited as very thin and irregularly shaped patches around the bases of the periodontal ligament fibers. Sharpey's fibers were short and poorly developed. In contrast, the development of the alveolar bone, the periodontal ligament, and the cellular cementum was seemingly unaffected. It is concluded that TNAP represents an essential factor in mantle dentin mineralization and in the formation of acellular cementum.

Correlations of genotype and phenotype in hypophosphatasia.

Hypophosphatasia, a rare inherited disorder characterized by defective bone mineralization, is highly variable in its clinical expression. The disease is due to various mutations in the tissue-non-specific alkaline phosphatase ( TNSALP ) gene. We report here the use of clinical data, site-directed mutagenesis and computer-assisted modelling to propose a classification of 32 TNSALP gene mutations found in 23 European patients, 17 affected with lethal hypophosphatasia and six with non-lethal hypophosphatasia. Transfection studies of the missense mutations found in non-lethal hypophosphatasia showed that six of them allowed significant residual in vitro enzymatic activity, suggesting that these mutations corresponded to moderate alleles. Each of the six patients with non-lethal hypophosphatasia carried at least one of these alleles. The three-dimensional model study showed that moderate mutations were not found in the active site, and that most of the severe missense mutations were localized in crucial domains such as the active site, the vicinity of the active site and homodimer interface. Some mutations appeared to be organized in clusters on the surface of the molecule that may represent possible candidates for regions interacting with the C-terminal end involved in glycosylphosphatidylinositol (GPI) attachment or with other dimers to form tetramers. Finally, our results show a good correlation between clinical forms of the disease, mutagenesis experiments and the three-dimensional structure study, and allowed us to clearly distinguish moderate alleles from severe alleles. They also confirm that the extremely high phenotypic heterogeneity observed in patients with hypophosphatasia was due mainly to variable residual enzymatic activities allowed by missense mutations found in the human TNSALP gene.

Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia. Mutations in brief no. 217. Online.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).

Expression of the mutant (1735T-DEL) tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients.

Hypophosphatasia (HOPS) is an inherited disorder characterized by defects in skeletal mineralization due to the deficiency of tissue-nonspecific alkaline phosphatase (TNSALP). To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at position 1735 (1735T-del) located in exon 12 has been detected in three genetically unrelated Japanese patients, which seems to be one of the hot spots among the causative mutations in Japanese HOPS patients. 1735T-del causes a frame shift downstream from codon 503 (Leu), and consequently the normal termination codon at 508 is eliminated. Since a new inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein is expected to have 80 additional amino acids. Expression of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP activity, and its molecule size was larger than normal, as expected. Interestingly, an immunoprecipitation study of patients' sera using antibody against TNSALP revealed an abnormal protein which corresponded in size to the mutated TNSALP expressed by COS-1 cells, suggesting that the abnormal TNSALP is made by HOPS patients. The detection of TNSALP in cells transfected with 1735T-del using an immunofluorescent method exhibited only a faint signal on the cell surface, but an intense intracellular fluorescence after permeabilization.

The effects of retinoic acid on alkaline phosphatase activity and tissue-non-specific alkaline phosphatase gene expression in human periodontal ligament cells and gingival fibroblasts.

Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.

Identification of fifteen novel mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in European patients with severe hypophosphatasia.

Hypophosphatasia is an inherited disorder characterised by defective bone mineralisation and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterisation of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 13 European families affected by perinatal, infantile or childhood hypophosphatasia. Eighteen distinct mutations were found, only three of which had been reported previously in North American and Japanese populations. Most of the 15 new mutations were missense mutations, but we also found two mutations affecting donor splice sites and a nonsense mutation. A missense mutation in the last codon of the putative signal peptide probably affects the final maturation of the protein. Despite extensive sequencing of the gene and its promotor region, only one mutation was identified in two cases, one of which was compatible with a possible dominant effect of certain mutations and the putative role of polymorphisms of the TNSALP gene. In 12 of the 13 tested families, genetic diagnosis was possible by characterisation of the mutations or by use of polymorphisms as genetic markers. Hypophosphatasia diagnosis was assigned in two families where clinical, laboratory and radiographic data were unclear and prenatal diagnosis was performed in one case. The results also show that severe hypophosphatasia is due to a very large spectrum of mutations in European populations with no prevalent mutation and that genetic diagnosis of the disease must be performed by extensive analysis of the gene.

Retinoic acid and methylation cis-regulatory elements control the mouse tissue non-specific alkaline phosphatase gene expression

To understand the mechanisms regulating the tissue non-specific alkaline phosphatase (TNAP) activity during development, we characterized cis-transcriptional regulatory elements. In embryonic cells and tissues, TNAP expression was driven preferentially by the exon 1A (E1A) promoter, one of the two promoters previously defined. Transcriptional activity of E1A promoter was up-regulated by retinoic acid (RA) through a putative RA-responsive element. Transgenic mice analysis with lacZ reporter constructs revealed negative regulatory elements within 8.5 kb of E1A promoter. Promoter sequences of endogenous TNAP in non-expressing tissues and those carried by the 8.5 kb- lacZ transgene were found to be highly methylated. A 1 kb fragment of E1A promoter increased the methylation level of lacZ and promoter sequences. The role of RA and DNA methylation in defining the embryonic expression pattern of TNAP is discussed.

Expression of tissue non‐specific alkaline phosphatase stimulates differentiated behaviour in specific transformed cell populations

Background Tissue non-specific alkaline phosphatase (TN-AP) is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic activity. This enzyme is distributed virtually in all mammalian tissues during embryonic development (it can be demonstrated as early as the 2-cell stage) where its expression is stage specific. The expression of TN-AP is frequently associated with cell differentiation and as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN-AP expression might influence cellular proliferation and morphological differentiation. The focus of this study was to determine whether this was a cell-specific effect or not. Methods The effects of TN-AP on various aspects of cellular activity were assessed by transferring and expressing the gene for this enzyme into three target populations; 1) CHO, 2) R1610, and 3) Rat-2. The parameters of cellular activity studied included cellular proliferation, cell cycle, cell migration, and tumorigenic potential (in the nude mouse). Cell cycle and cell proliferation analyses were accomplished, in part, through the use of fluorescence activated cell sorting (FACS) as were determinations of cell-associated TN-AP activity and amount. Northern and southern blot analyses were used to estimate gene copy number and to evaluate gene expression respectively in transfected cell lines. Results Our data indicate that the TN-AP gene under control of various gene promoters was stably integrated into three fibroblast-like cell lines (CHO, R1610, and Rat-2). TN-AP activity and TN-AP protein levels were correlated to the strength of the various gene promoters, but not to inserted gene copy numbers. The expression of the TN-AP gene in these three cell types further suggests cell-specific effects as demonstrated by the following findings. The expression of TN-AP under control of a weak gene promoter in CHO and Rat-2 cells clearly decreased cell proliferation and cell migration. However, the expression of TN-AP under control of either a weak or even a strong gene promoter in R1610 cells did not induce any changes in that cell line's behaviour (apart from expression of TN-AP). Such changes in CHO cells were also associated with a 2.2-fold increase in tubulin transcription as well as suppressed tumorigenic potential. Conclusion Taken together, these findings suggest that the inhibitory effects of TN-AP expression on proliferation and cell migration are not non-specific and that high expression of TN-AP may induce changes in cell behaviour which may be consistent with or at least related to induction of terminal differentiation. © 1996 Wiley-Liss, Inc.

Expression of tissue non-specific alkaline phosphatase stimulates differentiated behaviour in specific transformed cell populations.

Tissue non-specific alkaline phosphatase (TN-AP) is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic activity. This enzyme is distributed virtually in all mammalian tissues during embryonic development (it can be demonstrated as early as the 2-cell stage) where its expression is stage specific. The expression of TN-AP is frequently associated with cell differentiation and as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN-AP expression might influence cellular proliferation and morphological differentiation. The focus of this study was to determine whether this was a cell-specific effect or not. The effects of TN-AP on various aspects of cellular activity were assessed by transferring and expressing the gene for this enzyme into three target populations; 1) CHO, 2) R1610, and 3) Rat-2. The parameters of cellular activity studied included cellular proliferation, cell cycle, cell migration, and tumorigenic potential (in the nude mouse). Cell cycle and cell proliferation analyses were accomplished, in part, through the use of fluorescence activated cell sorting (FACS) as were determinations of cell-associated TN-AP activity and amount. Northern and southern blot analyses were used to estimate gene copy number and to evaluate gene expression respectively in transfected cell lines. Our data indicate that the TN-AP gene under control of various gene promoters was stably integrated into three fibroblast-like cell lines (CHO, R1610, and Rat-2). TN-AP activity and TN-AP protein levels were correlated to the strength of the various gene promoters, but not to inserted gene copy numbers. The expression of the TN-AP gene in these three cell types further suggests cell-specific effects as demonstrated by the following findings. The expression of TN-AP under control of a weak gene promoter in CHO and Rat-2 cells clearly decreased cell proliferation and cell migration. However, the expression of TN-AP under control of either a weak or even a strong gene promoter in R1610 cells did not induce any changes in that cell line's behaviour (apart from expression of TN-AP). Such changes in CHO cells were also associated with a 2.2-fold increase in tubulin transcription as well as suppressed tumorigenic potential. Taken together, these findings suggest that the inhibitory effects of TN-AP expression on proliferation and cell migration are not non-specific and that high expression of TN-AP may induce changes in cell behaviour which may be consistent with or at least related to induction of terminal differentiation.

Tissue Non-Specific Alkaline Phosphatase May Be a Function-Related Marker in Renal Proximal Tubular Epithelia and in Vascular Endothelia as It Is in Osteoblasts

Tissue non-specific alkaline phosphatase (TN-AP) is a multifunctional phosphatase and has a variety of substrates in vitro. In adults, TN-AP is highly expressed in osteoblasts, renal proximal tubular epithelia and vascular endothelia. In osteoblasts, it is well known that TN-AP plays an important role in bone mineralization. Considering its many activities in vitro, we hypothesize that TN-AP also plays a role in regulating cellular activities in renal proximal tubular epithelia and vascular endothelia. By using a stable gene transfection technique, we transfected two cell lines with a rat TN-AP gene, one renal proximal tubule-like (LLC-PK1), another vascular endothelia-like (Py-4-1). The effect of TN-AP expression was cell specific. In Py-4-1 cells, the forced TN-AP expression stimulates cellular behaviours associated with terminal differentiation. This suggests that in vivo AP expression in certain vascular endothelia may mark a terminal-differentiated subpopulation with less angiogenic potential. While, in LLC-PK1 cells, the forced TN-AP expression inhibited cellular transport function as demonstrated by delayed dome formation. By inference, TN-AP expression in renal proximal tubule in vivo should have a regulatory role in cellular transport. Our data suggest that TN-APs have roles as function-related markers in renal proximal tubular epithelia and vascular endothelia.

Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells

Mouse primordial germ cells express tissue non-specific alkaline phosphatase (TNAP) during development, but the widespread expression of another alkaline phosphatase gene in the early embryo limits the potential use of this marker to trace germ cells. To attempt to identify germ cells at all stages during embryonic development and to understand the role of TNAP in germ cell ontogeny, mice carrying a beta geo (lacZ/neor) disrupted allele of the TNAP gene were generated by homologous recombination in embryonic stem cells. Using beta-galactosidase activity, the embryonic pattern of TNAP expression was examined from the blastocyst stage to embryonic day 14. Results indicate that primordial germ cell progenitors do not express TNAP prior to gastrulation although at earlier times TNAP expression is found in an extraembryonic lineage destined to form the chorion. In homozygous mutants, primordial germ cells appear unaffected indicating that TNAP is not essential for their development or migration.

Changes in the pattern of expression of alkaline phosphatase in the mouse uterus and placenta during gestation

The development of the rodent chorio-allantoic placenta is a complicated process that results in the formation of a transport system capable of sustaining embryonic and fetal growth and development. Intimately linked to this process is alkaline phosphatase (AP), a cell-surface glycoprotein that possibly functions as a transport protein. In the present study, we have mapped the location of AP-expressing cells in the mouse utero-placental unit during the development of the chorio-allantoic placenta by use of enzyme histochemistry and in situ hybridization histochemistry. We found that at implantation the expression of the tissue non-specific AP (TNAP) gene is located exclusively in the decidua and that most of this decidual expression ceases as the placenta starts to form. One exception is a mesometrially located marginal zone of the decidua, which continues to express the TNAP gene until day 12 and the active protein until at least day 16. Trophoblasts of the chorion already express AP before the time of fusion with the ectoplacental cone, after which AP is expressed by trophoblasts of the resulting ectoplacental plate. AP expression in the mature chorio-allantoic placenta is localized in the placental labyrinth and spongy zones. In the latter zone, expression ceases on about day 14. Giant trophoblasts start to express AP on about day 10, with some cells still positive for AP at day 16. The yolk sac does not express AP at any developmental stage. The results show that AP expression during placental development is neither restricted to cells known to be involved in transport, nor expressed in all cells thought to be involved in this transport. This may indicate that AP is not merely a transport protein but has additional functions.

Hereditary hypophosphatasia and hyperphosphatasia

Hereditary hypophosphatasia and hyperphosphatasia are two rare skeletal disorders, which commonly present during childhood and are characterized by a decrease and an increase, respectively, in serum tissue nonspecific alkaline phosphatase activity. The primary defect in hereditary hypophosphatasia appears to be a structural abnormality in the tissue nonspecific alkaline phosphatase gene, resulting in a decrease or absence of enzymatic activity. The pathogenesis of hyperphosphatasia is unknown. There is no treatment for hypophosphatasia. Treatment with pamidronate appears promising for patients with hereditary hyperphosphatasia.

Missense Mutations of the Tissue-Nonspecific Alkaline Phosphatase Gene in Hypophosphatasia

Hypophosphatasia is an inborn error of metabolism that is characterized clinically by defective bone mineralization and biochemically by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) in serum and in tissues. Clinical severity is extremely variable, ranging from death in utero to pathologic fractures first presenting in adulthood. Severe forms of the disease are inherited in an autosomal recessive fashion; the modes of transmission of mild forms are uncertain. Deficiency of TNSALP activity in this condition suggests that mutations in the TNSALP "candidate" gene are the primary defects. This hypothesis was supported in 1988 by the demonstration, in one inbred infant, that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal hypophosphatasia. Here we summarize the work leading to that discovery and discuss the recent identification of additional missense mutations in the TNSALP gene associated with the entire clinical spectrum of hypophosphatasia.

Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia.

Hypophosphatasia is a heritable form of rickets/osteomalacia with extremely variable clinical expression. Severe forms are inherited in an autosomal recessive fashion; the mode of transmission of mild forms is uncertain. The biochemical hallmark of hypophosphatasia is deficient activity of the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Previously, we demonstrated in one inbred infant that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal disease. We have now examined TNSALP cDNAs from four unrelated patients with the severe perinatal or infantile forms of hypophosphatasia. Each of the eight TNSALP alleles from these four individuals contains a different point mutation that causes an amino acid substitution. These base changes were not detected in at least 63 normal individuals and, thus, appear to be causes of hypophosphatasia in the four patients. (Two additional base substitutions, found in one allele from each of the four patients, are linked polymorphisms.) Twenty-three unrelated patients (of 50 screened), who reflect the entire clinical spectrum of hypophosphatasia, possess one of our of the above eight mutations. In two of these additional patients, mild forms of the disease are also inherited in an autosomal recessive fashion. Our findings indicate that hypophosphatasia can be caused by a number of different missense mutations and that the specific interactions of different TNSALP mutant alleles are probably important for determining clinical expression. Severe forms, perinatal and infantile disease, are largely the result of compound heterozygosity for different hypophosphatasia alleles. At least some cases of childhood and adult hypophosphatasia are inherited as autosomal recessive traits.