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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 2 743-747
Copyright © 2000 by The Endocrine Society

Original Studies

Asp361Val Mutant of Alkaline Phosphatase Found in Patients with Dominantly Inherited Hypophosphatasia Inhibits the Activity of the Wild-Type Enzyme

Hermann L. Müller, Miwa Yamazaki, Toshimi Michigami, Takashi Kageyama, Eckhard Schönau, Peter Schneider and Keiichi Ozono

Departments of Pediatrics (H.L.M.) and Nuclear Medicine (P.S.), University Hospital, D-97080 Würzburg; and the Department of Pediatrics, University Hospital (E.S.), 50924 Köln, Germany; and the Department of Environmental Medicine, Osaka Medical Center and Institute for Maternal and Child Health (M.Y., T.M., T.K., K.O.), Izumi, Osaka 594-1101, Japan

Address all correspondence and requests for reprints to: Keiichi Ozono, M.D., Department of Environmental Medicine, Osaka Medical Center and Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypophosphatasia is characterized by the hypomineralization of bone associated with the mutation of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. Although the disease is usually autosomal recessive, an autosomal dominant form is also recognized. Approximately 50 mutations have been found in the TNSALP gene in patients with hypophosphatasia. However, the mutations identified to date do not seem to account for the dominantly inherited form of the disease. We have examined a German family in which the father and all 4 children were affected with hypophosphatasia, whereas the mother was healthy. The affected members of this family showed premature loss of deciduous teeth at or shortly before 2 yr of age and low levels of serum ALP with elevated levels of urinary phosphoethanolamine. DNA analysis by direct sequencing revealed a heterozygous missense mutation that caused the conversion of amino acid Asp to Val at position 361 (D361V) in the patients. Another substitution was detected in exon 12 (Val to Ala conversion at codon 505: V505A) in 1 allele of the mother and 3 children, indicating no association of the substitution with the disease. Reconstruction experiments demonstrated that the D361V mutant protein lost its enzymatic activity and that it inhibited the function of wild-type enzyme when coexpressed in COS-7 cells. On the other hand, the V505A mutant exhibited enzymatic activities equal to those of the wild-type ALP. It is likely that the mutant D361V protein forms dimers with the wild-type protein, and the protein-protein interaction contributes to the dominant effect of the mutant D361V. The mutation that causes D361V is the first one proven to be associated with the dominant form of hypophosphatasia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPOPHOSPHATASIA, which is characterized by the hypomineralization of bone associated with the impaired activity of tissue-nonspecific alkaline phosphatase (TNSALP), is divided into five subclasses: perinatal, infantile, childhood, and adult type, based on the age at clinical presentation, and odontohypophosphatasia, where only dental manifestations are noted (1). The disease is usually transmitted in an autosomal recessive manner (McKusick 241500, 241510); however, autosomal dominant inheritance is also recognized in mild forms or adult-type hypophosphatasia (McKusick 146300). To consider the type of inheritance, we have to be careful to distinguish between a patient with a mild form of hypophosphatasia and a carrier of autosomal recessive hypophosphatasia (1, 2, 3).

The TNSALP gene consists of 12 exons, and the coding exons are 2–12 (4). Approximately 50 different mutations have been identified in the TNSALP gene, and most of them were missense mutations (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Reported mutations associated with hypophosphatasia are scattered throughout the coding exons. The elucidation of the molecular heterogeneity underlying hypophosphatasia may contribute to our understanding of the clinical heterogeneity observed in this disorder. For example, mutations at nucleotides 747, 1057, and 1309 are associated with mild (childhood and adult-type) hypophosphatasia (18). In addition, mutagenesis experiments revealed that the enzymatic activity of alkaline phosphatase (ALP) mutants seems to correlate with the severity of the disease to some extent (17). However, further analysis of the function of the mutated ALP is necessary to determine a genotype-phenotype correlation, especially for the mild form, because the enzymatic activity of ALP protein may differ in vivo and in vitro.

Although we can speculate that the mutations responsible for the dominantly inherited hypophosphatasia inhibit the enzymatic activity of wild-type ALP, no study has proved the dominant negative effect of mutated ALP to date. It is known that ALP exhibits enzymatic activity by forming dimer or tetramer complexes. Therefore, the mutant ALP found in hypophosphatasia with dominant inheritance may suppress the wild-type ALP activity through multimer formation. A similar type of mechanism has been reported for osteogenesis imperfecta, generalized thyroid hormone resistance, long QT syndrome, and others (19, 20, 21).

In the present report we describe a family with an autosomal dominant form of hypophosphatasia and the study of the TNSALP gene in all family members. The mutation (Asp361Val) of the TNSALP gene was found to be heterozygous in affected members. The mutation caused not only loss of enzymatic activity of the ALP enzyme, but also inhibition of the function of wild-type ALP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Family information

The father of the family (38 yr old, Caucasian) had premature loss of deciduous teeth shortly before 2 yr of age. He complained of the onset of low back pain at the age of 24 yr. The pain became chronic, involving the lower extremities, and exacerbated during physical activity despite all forms of conservative therapy. Neurological deficits or reflex pathologies were not found. A magnetic resonance imaging study of the knees revealed a 2-cm area of focal marrow edema in the lateral femoral condyle, which was interpreted as an early sign of osteoarthritis. A whole body 99mTc bone scintigraphy demonstrated symmetric foci of increased activity in the patello-femoral joints, consistent with mild degenerative osteoarthropathy. Reduced serum ALP activity and elevated phosphoethanolamine (PEA) excretion led to the diagnosis of hypophosphatasia of the adult type in the father (Table 1Go and Fig. 1Go).


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  		Table 1. Parameters of bone metabolism and bone density measurements in the children and parents

 


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  		Figure 1. Pedigree of the family. Serum ALP activities of members are described; {downarrow} shows the lower than reference values of the age-matched subjects. *, Premature loss of deciduous teeth. Hatched and shaded symbols represent Asp361Val and Val505Ala mutations of TNSALP gene, respectively. Nucleotides at four indicated positions of each allele are also shown in boxes.

 
All four children of the father (one male and three females) had premature loss of deciduous teeth at or shortly before 2 yr of age as an onset of symptoms. The history of dentition in the mother (36 yr old, Caucasian, no cosanguinity) and the maternal grandparents was reported to be normal. None of the children presented with a history of skeletal pain, reduced growth rate, or pathological findings on physical examination. All members of the family except the mother showed levels of ALP activity below normal and markedly elevated PEA excretion in the urine (Table 1Go and Fig. 1Go). The maternal grandmother had levels within the normal range for ALP, PEA, and inorganic phosphorus. The paternal grandfather and grandmother are not known, as the father is an adoptee. Skeletal roentgenograms, bone density measurements, and biochemical analysis of bone metabolism revealed no pathological findings, except decreased activity of ALP in the children (Table 1Go). A review of the dental records and thorough dental examination, including histological examination of nonprematurely lost primary teeth, showed no abnormalities.

General methods

Serum was obtained by centrifugation of whole blood samples and was frozen within 2 h of collection. Unless otherwise stated, biochemical analyses of serum and urine were performed using commercial assay kits. The isoforms of ALP were quantified after electrophoretic separation on titan gels using a commercial assay (Helena Diagnostika, Sunderland, Great Britain). Urinary PEA excretion and serum concentrations of PEA and procollagen 1 peptide were determined in all four children using methods described previously (22) (Table 1Go). Type 1 procollagen was measured using a commercial RIA (Orion, Espoo, Finland).The cross-link, deoxypyridinoline, was quantified using a chemiluminescent enzyme immunoassay (Diagnostic Products, Los Angels, CA). Bone density was estimated by peripheral quantitative computed tomography (Stratec Medizintechnik, Pforzheim, Germany) (23).

Sequence analysis. DNA sequencing was performed after extraction of genomic DNA from the peripheral mononuclear cells. Exons 2–12 were amplified using PCR primers previously described (14). The fragments amplified by PCR were sequenced directly or after the subcloning into pT7-Blue T-vector (Novagen, Madison, WI), using a model 377 sequencer (Perkin-Elmer Corp., Norwalk, CT) and the BigDye terminator technology (Perkin-Elmer Corp.). The nucleotide and amino acid numbers are designated relative to the initiation of complementary DNA (cDNA) and the mature enzyme, respectively (24).

Mutagenesis and expression. Mutagenesis of the substitution of A at nucleotide 1309 for T, leading to the Asp361Val mutation (D361V), was achieved by the Kunkel method following the manufacturer’s recommendations (Gene Editor, Promega Corp., Madison, WI). Mutagenesis of the T to C conversion at nucleotide 1741, leading to the Val505Ala mutation (V505A), was achieved by PCR-mediated mutagenesis as reported previously (12). The human TNSALP cDNA was provided in the plasmid, pSV2Aalp, by Dr. P. S. Henthorn (University of Pennsylvania, Philadelphia, PA), and an expression vector was constructed by the insertion of the cDNA into pcDNA3 vector (Invitrogen, Carlsbad, CA) (24). The expression vectors of the normal and mutated ALP cDNA were transfected into COS-7 cells by the lipofection method (Lipofectamine, Life Technologies, Inc., Grand Island, NY). The enzymatic activity of ALP was measured by the Lowry method, using p-nitrophenylphosphate as a substrate, 48 h after the transfection and was normalized with the amount of protein present (25).

Construction of the fusion proteins of ALP and green fluorescent protein (GFP). To distinguish wild-type ALP from mutants, we generated the fusion proteins of TNSALP and GFP-ALP. Expression vectors were reconstructed in pcDNA3.1 vector (Invitrogen) using synthesized nucleotides coding the signal peptide of TNSALP, GFP cDNA (238 amino acids), obtained by digestion of pGreen Lantern (Life Technologies, Inc.) with NotI, and the cDNA of mature TNSALP (507 amino acids in wild-type ALP) obtained from pSV2Aalp. In transient transfection experiments, the fusion proteins encoding expression vectors of wild-type (pcDNA-sig-GFP-ALP) and/or the same amount of plasmids for mutated ALPs without GFP (ALPD361V and ALPV505A) were transfected into COS-7 cells grown in a 60-mm dish using Lipofectamine (Life Technologies, Inc.). Forty-eight hours after the transfection, cells were lysed in 200 µl RIPA buffer [1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 5 mM ethylenediamine tetraacetate, and protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany)]. The fusion proteins were immunoprecipitated using monoclonal anti-GFP antibody (Roche Molecular Biochemicals) at 4 C for 3 h, followed by the incubation with protein A/G-conjugated agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C overnight. The agarose on which the immunocomplex was immobilized was washed with RIPA buffer and aliquoted. The aliquot of each sample was used for the ALP assay. An aliquot of the same sample was used for Western analysis using the mouse monoclonal anti-GFP antibody (Roche Molecular Biochemicals). After incubation with peroxidase-labeled antimouse IgG antibody (Amersham Pharmacia Biotech, Aylesbury, UK), the immunocomplex was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutation analysis of the TNSALP gene

A missense mutation (A to T conversion at nucleotide 1309 of the cDNA) was detected in exon 10 (Asp to Val conversion at codon 361: D361V) in one allele of all affected members (father and all four children). An additional mutation (T to C conversion at nucleotide 1741 of the cDNA) was also found in exon 12 (Val to Ala conversion at codon 505: V505A) in one allele of the mother and three of the children. No other mutation was found in the entire coding region of the TNSALP cDNA.

Polymorphisms due to single base substitutions in the coding region

Two previously described polymorphisms in the TNSALP gene were also analyzed in the family members (18). The bases of the reported polymorphic site in exon 7 (at nucleotide 963) and exon 9 (at nucleotide 1052) were analyzed. The father was heterozygous in these sites (T/C and A/G, respectively), and the mother was homozygous (T and A, respectively). All four children were heterozygous, and these results together with the mutations described above determine the linkage of nucleotides at all four sites (nucleotide at 963, 1052, 1309, and 1741). T responsible for the D361V mutation was located on the allele that contained C, G, and T at nucleotides 963, 1052, and 1741, respectively. Genotypes of all members were as follows (Fig. 1Go): father: CGTT, TAAT; mother: TAAT, TAAC; three of the children: CGTT, TAAC; and the other child: CGTT, TAAT. Each of these four letters is with first set of letters representing nucleotides at position 963, 1052, 1309, and 1741, respectively.

Expression of the mutated cDNA of the ALP

The activity of the mutated ALP enzymes was measured using the transient expression system. The COS-7 cells transfected with the mutated plasmid at nucleotide 1309 (D361V) exhibited low ALP activity, similar to the COS-7 cells transfected with the mock expression vector (25.4 ± 4.8% and 30.2 ± 5.6%, respectively). In contrast, the COS-7 cells transfected with the mutated cDNA at codon 505 (V505A) exhibited a level 94.2 ± 16.9% of the wild-type ALP control level (Fig. 2Go).



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  		Figure 2. The activity of ALP exhibited in COS-7 cells transfected with the expression vector. The expression vector of wild-type TNSALP or mutants (D361V and V505A) was generated as described in Materials and Methods and introduced into COS-7 cells. Two days after the transfection, the ALP enzymatic activity of the cell lysates was evaluated using p-nitrophenylphosphate as substrate. The enzymatic activity of the wild-type TNSALP is taken as 100%. The data are expressed as the mean ± SE.

 
To investigate the effect of the ALP mutant on the enzymatic activity of wild-type ALP, the activity was evaluated after the introduction of expression vectors of both wild-type and mutated ALPs. The coexpression of the ALPD361V mutant inhibited the enzymatic activity of wild-type ALP in a dose-dependent manner (Fig. 3Go). The dominant negative effect was also observed in the other expression vector, pcDNA-sig-GFP-ALP, with pcDNA-ALPD361V after the immunoprecipitation (Fig. 4AGo). The protein level of wild-type ALP was estimated as a GFP-tagged protein in the Western blot using an anti-GFP antibody. Coexpression of ALPD361V did not disturb the protein level of wild-type ALP with GFP based on the Western blot analysis using an anti-GFP antibody (Fig. 4BGo).



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  		Figure 3. The suppression of enzymatic activity of wild-type TNSALP by D361V mutants. The expression vectors of wild-type TNSALP and/or D361V mutants were introduced into COS-7 cells. The amount of each expression vector is given at the bottom. The total amount of vectors was the same among groups after addition of the mock vector. The enzymatic activity of the wild-type TNSALP is taken as 100%. The data are expressed as the mean ± SE.

 


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  		Figure 4. Analysis of the suppressive effect of D361V by adjusting the expression level of TNSALP. The expression vector of GFP-tagged wild-type TNSALP and/or untagged D361V mutants was introduced into COS-7 cells. Three days after transfection, the cell lysates were immunoprecipitated with anti-GFP monoclonal antibody. Some of the immunoprecipitates were used to estimate the enzymatic activity (A), and the rest were used in Western blotting (B) with a mouse monoclonal anti-GFP antibody. After incubation with peroxidase-labeled antimouse IgG antibody, the immunocomplex was visualized by enhanced chemiluminescence.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hypophosphatasia is characterized by the hypomineralization of bone associated with the impaired activity of TNSALP. However, it is sometimes difficult to diagnose mild forms of hypophosphatasia, especially to distinguish a patient from a carrier. In such cases, premature loss of decidual teeth is a hallmark of the disease, and increased levels of urinary PEA and decreased levels of serum pyridoxal-5'-phosphate are suggestive of the disease (26).

In the family described here, the father and all four children, consisting of a boy and three girls, exhibited premature loss of decidual teeth, low activity of serum ALP, and elevated levels of urinary PEA. In contrast, the mother and her mother had no symptoms related to the disease and exhibited normal serum ALP and urinary PEA levels. These results show that the father and all children were affected with a mild form of hypophosphatasia, consistent with other reported cases of dominant inheritance, whereas the mother was a healthy noncarrier. This suggests that hypophosphatasia is inherited in an autosomal dominant manner in this family.

Analysis of the TNSALP gene in the family revealed that the D361V mutation existed heterozygously in the father and in all of the children, but not in the mother. Another mutation that caused V505A was found in the mother and three of the children, but not in the father and the other child, who were affected with the disease. These results suggest that the D361V, not the V505A, mutation is responsible for the disease.

Consistent with our hypothesis, reconstruction experiments proved that the D361V mutant lost its enzymatic activity, whereas the level of activity in the V505A mutant was comparable with that in the wild-type protein. These results demonstrate that D361V is the cause of dominant inherited hypophosphatasia in this family and that V505A is a polymorphism, although we did not investigate the rate of V505A in the normal population.

To test our hypothesis, COS-7 cells were cotransfected with the expression vector of the mutant and wild-type ALP to analyze the difference between the usual recessive and the dominant traits. It was found that the D361V mutant inhibited the enzymatic activity of wild-type ALP in a dose-dependent manner, i.e. the D361V mutant acted in a dominant negative fashion. These results clearly illustrated the molecular basis of the dominant inheritance of hypophosphatasia. To exclude the possibility that the reduction in the expression of the wild-type ALP was caused by coexpression of D361V mutant, we determined the amount of protein by Western blotting after constructing a fusion protein of ALP with GFP and ALP activity. The results showed that the D361V mutant suppressed wild-type ALP activity as well in this construct, and that the protein level was not affected by coexpression of D361V. As ALP functions through forming dimer or tetramer complexes, it is likely that D361V influenced the activity of wild-type ALP by protein-protein interaction. However, the mechanism by which D361V mutant exerted the dominant negative effect remains to be elucidated. Actually, the D361V mutation was suggested to be a cause of dominant hypophosphatasia (27), but there had been no thorough study performed on it to our knowledge. We confirmed that the D361V mutation is responsible for the dominantly inherited form of hypophosphatasia by showing a dominant negative effect of the D361V mutant. The expression level of the D361V mutant determines the degree of inhibition of the function of wild-type ALP, and this may affect the penetrance of the disease. Our study lays the basis for future investigations into the phenotype-genotype correlation in hypophosphatasia.

In conclusion, we described a family with an autosomal dominant form of hypophosphatasia and the study of the TNSALP gene in all family members. The mutation (D361V) of the TNSALP gene was found to exist heterozygously in affected members. The mutant protein not only loses its own enzymatic activity, but also interferes with the function of wild-type ALP.


   Acknowledgments
 
We thank Dr. P. S. Henthorn of the University of Pennsylvania School of Veterinary Medicine for generously providing the TNSALP expression vector. We also thank Ms. Tomoko Hayashi for secretarial assistance.

Received July 13, 1999.

Revised October 18, 1999.

Accepted October 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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