Perinatal hypophosphatasia: Radiology,pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)‐related defective bone mineralization due to mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA‐based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term. © 2001 Wiley‐Liss, Inc.     

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).     

Matrix vesicles in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals.

Hypophosphatasia, a heritable disease characterized by deficient activity of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP), results in rickets and osteomalacia. Although identification of TNSALP gene defects in hypophosphatasia establishes a role of ALP in skeletal mineralization, the precise function remains unclear. The initial site of mineralization (primary mineralization) normally occurs within the lumen of TNSALP-rich matrix vesicles (MVs) of growth cartilage, bone, and dentin. We investigated whether defective calcification in hypophosphatasia is due to a paucity and/or a functional failure of MVs secondary to TNSALP deficiency. Nondecalcified autopsy bone and growth plate cartilage from five patients with perinatal (lethal) hypophosphatasia were studied by nondecalcified light and electron microscopy to assess MV numbers, size, shape, and ultrastructure and whether hypophosphatasia MVs contain apatite-like mineral, as would be the case if these MVs retained their ability to concentrate calcium and phosphate internally despite a paucity of TNSALP in their investing membranes. We found that hypophosphatasia MVs are present in approximately normal numbers and distribution and that they are capable of initiating internal mineralization. There is retarded extravesicular crystal propagation. Thus, in hypophosphatasia the failure of bones to calcify appears to involve a block of the vectorial spread of mineral from initial nuclei within MVs, outwards, into the matrix. We conclude that hypophosphatasia MVs can concentrate calcium and phosphate internally despite a deficiency of TNSALP activity.     

Evidence of a founder effect for the tissue-nonspecific alkaline phosphatase (TNSALP) gene E174K mutation in hypophosphatasia patients

Hypophosphatasia is a rare inborn error of metabolism characterised by defective bone mineralisation caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The clinical expression of the disease is highly variable, ranging from stillbirth with a poorly mineralised skeleton to pathologic skeletal fractures which develop in late adulthood only. This clinical heterogeneity is due to the strong allelic heterogeneity in the TNSALP gene. We found that mutation E174K is the most frequent in Caucasian patients, and that it was carried by 31% of our patients with mild hypophosphatasia. Because the mutation was found in patients of various geographic origins, we investigated whether it had a unique origin or rather multiple origins due to recurrence of de novo mutations. Three intragenic polymorphisms, S93S, 472+12delG and V505A, were genotyped in patients carrying E174K and in normal unrelated individuals. Our results show that all the E174K mutations are carried by a common ancestral haplotype, also found at low frequency in normal and hypophosphatasia chromosomes. We conclude that the TNSALP gene E174K mutation is the result of a relatively ancient ancestral mutation that occurred on a single chromosome in the north of Western Europe and spread throughout the rest of Europe and into the New World as a result of human migration.     

Denaturing gradient gel electrophoresis analysis of the tissue nonspecific alkaline phosphatase isoenzyme gene in hypophosphatasia

Hypophosphatasia, a heritable form of rickets/osteomalacia, was first described in 1948. The biochemical hallmark, subnormal alkaline phosphatase (ALP) activity in serum, reflects a generalized disturbance involving the tissue-nonspecific isoenzyme of ALP (TNSALP). Deactivating mutations in the gene that encodes TNSALP have been reported in patients worldwide. Nevertheless, hypophosphatasia manifests an extraordinary range of clinical severity spanning death in utero to merely premature loss of adult teeth. There is no known medical treatment. To delineate the molecular pathology which explains the disease variability and to clarify the pattern(s) of inheritance for mild cases of hypophosphatasia, we developed comprehensive mutational analysis of TNSALP. High efficiency of mutation detection was possible by denaturing gradient gel electrophoresis (DGGE). Primers and conditions were established for all TNSALP coding exons (2-12) and adjacent splice sites so that the amplicons incorporated a GC clamp on one end. For each amplicon, the optimum percentage denaturant was determined by perpendicular DGGE. In 19 severely affected pediatric subjects (having perinatal or infantile hypophosphatasia or early presentation during childhood) from among our large patient population, we detected 2 TNSALP mutations each in 16 patients (84%) as expected for autosomal recessive disease. For 2 patients (11%), only 1 TNSALP mutation was detected by DGGE. However, one subject (who died from perinatal hypophosphatasia) had a large deletion as the second mutation. In the other (with infantile hypophosphatasia), no additional mutation was detected by DNA sequencing of all protein-coding exons. Possibly, she too has a deletion. For the final patient, with unclassifiable hypophosphatasia (5%), we detected only a single mutation which has been reported to cause relatively mild autosomal dominant disease; the other allele appeared to be intact. Hence, DGGE analysis was 100% efficient in detecting mutations in the coding exons and adjacent splice sites of TNSALP in this group of severely affected patients but, as expected, failed to detect a large deletion. To date, at least 78 different TNSALP mutations (in about 70 hypophosphatasia patients) have been reported globally. In our large subset of severely affected patients, we identified 8 novel TNSALP mutations (Ala34Ser, Val111Met, Delta G392, Thr117His, Arg206Gln, Gly322Arg, Leu397Met, and Gly409Asp) and 1 new TNSALP polymorphism (Arg135His) furthering the considerable genotypic variability of hypophosphatasia.     

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.     

A novel missense mutation of the tissue-nonspecific alkaline phosphatase gene detected in a patient with hypophosphatasia

Hypophosphatasia is a rare heritable inborn error of metabolism characterized by abnormal bone mineralization associated with a deficiency of alkaline phosphatase. The clinical expression of hypophosphatasia is highly variable, ranging from death in utero to pathologic fractures first presenting in adulthood. We investigated the tissue-nonspecific alkaline phosphatase (TNSALP) gene from a Japanese female patient with hypophosphatasia. By a quantitative polymerase chain reaction (PCR) method, the amount of TNSALP mRNA appeared to be almost equal to that in normal individuals. Gene analysis clarified that the hypophosphatasia originated from a missense mutation and a nucleotide deletion. The missense mutation, a C ? T transition at position 1041 of cDNA, results in an amino acid change from Leu to Phe at codon 272, which has not yet been reported. The previously reported deletion of T at 1735 causes a frame shift mutation downstream from Leu at codon 503. Family analysis showed that the mutation 1041T and the deletion 1735T had been inherited from the proband's father and mother, respectively. An expression experiment revealed that the mutation 1041T halved the expression of alkaline phosphatase activity. Using homology analysis, the Leu-272 was confirmed to be highly conserved in other mammals.     

Prevalence of c.1559delT in ALPL, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japanese and effects of the mutation on heterozygous carriers

Hypophosphatasia (HPP) is an inherited disorder caused by mutations in ALPL that encodes an isozyme of alkaline phosphatase (ALP), TNSALP. One of the most frequent ALPL mutations is c.1559delT, which causes the most severe HPP, the perinatal (lethal) form (pl-HPP). c.1559delT has been found only in Japanese and its prevalence is suspected to be high; however, the allele frequency of c.1559delT in Japanese remains unknown. We designed a screening system for the mutation based on high-resolution melting curve analysis, and examined the frequency of c.1559delT. We found that the c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1562-1/284). This indicates that ～1 in 900?000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. In our analysis, the majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine phosphoethanolamine. Our results indicate that the only way to reliably detect whether individuals are pl-HPP carriers is to perform the ALPL mutation analysis.     

First identification of a gene defect for hypophosphatasia: evidence that alkaline phosphatase acts in skeletal mineralization

Hypophosphatasia is a heritable disorder characterized by defective osteogenesis and deficient liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Severe forms of the disease are inherited in an autosomal recessive fashion. We examined cultured skin fibroblasts from twelve patients with severe hypophosphatasia. All were deficient in L/B/K ALP activity, yet produced normal levels of the corresponding mRNA. Sequence analysis of L/B/K ALP cDNA isolated from one of the patient-derived fibroblast lines revealed a point mutation that converted amino acid 162 of mature L/B/K ALP from alanine to threonine. The patient was homozygous and the parents, who are second cousins, heterozygous for this mutation. Introduction of the mutation into an otherwise normal cDNA disrupted the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene resulted in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.     

A case of lethal hypophosphatasia providing new insights into the perinatal benign form of hypophosphatasia and expression of the ALPL gene

Hypophosphatasia is a rare inherited bone disease caused by mutations in the alkaline phosphatase liver-type gene (ALPL) gene, with extensive allelic heterogeneity leading to a range of clinical phenotypes. We report here a patient who died from severe lethal hypophosphatasia, who was compound heterozygous for the mutation c.1133A>T (D361V) and the newly detected missense mutation c791A>G, and whose parents were both healthy. Because the c.1133A>T (D361V) mutation was previously reported to have a dominant-negative effect and to be responsible for the uncommon perinatal benign form of the disease, we studied the expression of the ALPL gene in this family. Analysis at the messenger RNA (mRNA) level, both quantitative and qualitative, showed that the paternal c.1133A>T (D361V) mutation was associated with over-expression of the ALPL gene and that the maternal c.791A>G mutation lead to complete skipping of exon 7. The results provide an explanation of the lethal phenotype in the patient where the two ALPL alleles are non-functional and in the asymptomatic father where over-expression of the normal allele could counteract the effect of the c.1133A>T (D361V) mutation by providing an increased level of normal mRNA. This may also explain the variable expression of hypophosphatasia observed in parents of patients with the perinatal benign form.     

First Identification of a Gene Defect for Hypophosphatasia: Evidence That Alkaline Phosphatase Acts in Skeletal Mineralization

Hypophosphatasia is a heritable disorder characteriazed by defective osteogenesis and deficient liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Severe forms of the disease are inherited in an autosomal recessive fashion. We examined cultured skin fibroblasts from twelve patients with severe hypophosphatasia. All were deficient in L/B.K ALP activity, yet produced normal levels of the corresponding mRNA. Sequence analysis of L/B/K ALP cDNA isolated from one of the patient-derived fibroblast lines revealed a point mutation that converted amino acid 162 of mature L/B/K ALP from alanine to threonine. The patient was homozygous and the parents, who are second cousins, heterozygous for this mutation. Introduction of the mutation into an otherwise normal cDNA disrupted the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene resulted in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.     

Asfotase Alfa in Perinatal/Infantile-Onset and Juvenile-Onset Hypophosphatasia: A Guide to Its Use in the USA

Subcutaneous asfotase alfa (Strensiq?), a first-in-class bone-targeted human recombinant tissue-nonspecific alkaline phosphatase (TNSALP) replacement therapy, is approved in the USA for the treatment of patients with perinatal/infantile- or juvenile-onset hypophosphatasia (HPP). In clinical trials, asfotase alfa was an effective and generally well tolerated treatment for perinatal/infantile- and juvenile onset-HPP through at least 3 and 5 years’ treatment, respectively. Relative to untreated age-matched, juvenile-onset-HPP historical control cohorts, survival and ventilation-free survival were significantly prolonged in asfotase alfa-treated patients, consequent to preceding improvements in bone mineralization.     

Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.     

Hypophosphatasia: the mutations in the tissue-nonspecific alkaline phosphatase gene

Hypophosphatasia is an inborn error of metabolism caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. Most of the 65 distinct mutations described to date are missense mutations, a result which must be correlated with the great variability of clinical expression ranging from stillbirth without mineralized bone to pathologic fractures developing only late in adulthood. Correlations of genotype and phenotype have been established on the basis of clinical data exhibited by the patients, transfection studies, computer-assisted modeling, and examination of biochemical properties of ALP in cultured fibroblasts of patients. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia, and screening may also be helpful in confirming diagnosis of hypophosphatasia when biochemical and clinical data are not clear. Screening is also the necessary first step in further studies to elucidate dominant transmission of the disease and of liver-, bone- and kidney-type alkaline phosphatase activity mechanism.     

Enhancement of drug delivery to bone: characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide

Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.     

Molecular effects of the tissue-nonspecific alkaline phosphatase gene polymorphism (787T > C) associated with bone mineral density

Based on studies of hypophosphatasia, which is a systemic skeletal disorder resulting from tissuenonspecific alkaline phosphatase (TNSALP) deficiency, TNSALP was suggested to be indispensable for bone mineralization. Recently, we demonstrated that there was a significant difference in bone mineral density (BMD) among haplotypes, which was lowest among TNSALP (787T [Tyr-246Tyr]) homozygotes, highest among TNSALP (787T > C [Tyr246His]) homozygotes, and intermediate among heterozygotes. To analyze protein translated from the TNSALP gene 787T > C, we performed the biosynthesis of TNSALPs using TNSALP cDNA expression vectors. TNSALP (787T) and TNSALP (787T > C) were synthesized similarly as a high-mannose-type 66-kDa form, becoming an 80-kDa form. Expression of the human 787T > C TNSALP gene using the cultured mouse marrow stromal cell line ST2 demonstrated that the protein translated from 787T > C exhibited an ALP-specific activity similarly to that of 787T. Interestingly, the Km value for TNSALP in ST2 cells transfected with the 787T > C TNSALP gene was decreased significantly compared to that of cells carrying the 787T gene (P < 0.01). These results suggest that the significant difference in Km values between the proteins translated from 787T > C and 787T may contribute to regulatory effects on bone metabolism.     

Perinatal hypophosphatasia: diagnosis and detection of heterozygote carriers within the family

We report on two families in which one or two children had a severe disorder of skeletal development detected by prenatal ultrasonography. The children died postnatally and showed typical radiological and biochemical findings of perinatal hypophosphatasia. Biochemical analysis revealed a low activity of alkaline phosphatase (AP) and a high value of pyridoxal-5-phosphate (PLP), one of its natural substrates. The screening for mutations of the tissue nonspecific alkaline phosphatase (TNSALP) gene showed homozygosity for a point mutation (G 317 --> D) in the two affected children of the first family. The affected child of the second family was homozygous for a nonsense mutation (R 411 --> X). Family screening revealed that the determination of AP and PLP is helpful for detection of heterozygotes. However, heterozygote children had values of AP in the lower normal range during phases of rapid growth. The determination of PLP proved to be more sensitive in these cases. It should be kept in mind that during the last trimester of gestation there is an increase in maternal AP activity and a normalization of PLP due to placental AP, which is not affected. Therefore, in the course of a prenatal diagnosis in an index case, paternal blood should be analyzed in parallel. For detailed genetic counseling and early prenatal diagnosis in following pregnancies, the possibility of mutation analysis should be used.     

Utility of genetic testing for prenatal presentations of hypophosphatasia

Hypophosphatasia (HPP) is a rare inherited disease affecting bone and dental mineralization due to loss-of-function mutations in the ALPL gene encoding the tissue nonspecific alkaline phosphatase (TNSALP). Prenatal benign HPP (PB HPP) is a rare form of HPP characterized by in utero skeletal manifestations that progressively improve during pregnancy but often still leave symptoms after birth. Because the prenatal context limits the diagnostic tools, the main difficulty for clinicians is to distinguish PB HPP from perinatal lethal HPP, the most severe form of HPP.We previously attempted to improve genotype phenotype correlation with the help of a new classification of variants based on functional testing. Among 46 perinatal cases detected in utero or in the neonatal period for whose ALPL variants could be classified, imaging alone was thought to clearly diagnose severe lethal HPP in 35 cases, while in 11 cases, imaging abnormalities could not distinguish between perinatal lethal and BP HPP. We show here that our classification of ALPL variants may improve the ability to distinguish between perinatal lethal and PB HPP in utero.     

Histologic and ultrastructural studies on the mineralization process in hypophosphatasia

Chondroosseous tissue from six infants with infantile hypophosphatasia and six control infants were studied by light, transmission, and scanning electron microscopy. Alkaline phosphatase histochemical reaction of the growth plate was studied in two infants and was greatly reduced when compared to two control infants. Hypertrophic chondrocytes were increased in number with persisting cartilage islets in the metaphysis. In five of the six cases studied, chondrocytes and intercartilagenous intercellular chondroid matrix appeared ultrastructurally normal. Matrix vesicle distribution was similar to that of control subjects, but they were associated with few mineral crystals. In two infants, the matrix vesicles were alkaline phosphatase nonreactive. In the calcifying zone of the growth plate and in the newly formed metaphyseal trabecular bone, cartilagenous calcospherites often were small and the orientation of crystals was nonradial when compared to that of control infants. The mineralization of diaphyseal bone appeared normal. It seems that matrix vesicles are present in hypophosphatasia and that the impaired mineralization of cartilage is due primarily to the deficiency of alkaline phosphatase. In spite of the lack of alkaline phosphatase, secondary mineralization of bone which is not mediated by matrix vesicles was normal.