Journal of Clinical Endocrinology & Metabolism
, doi:10.1210/jc.2004-1456
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2436-2439
Copyright © 2005 by The Endocrine Society
Childhood Hypophosphatasia Due to a de Novo Missense Mutation in the Tissue-Nonspecific Alkaline Phosphatase Gene
A. Taillandier,
S.-L. Sallinen,
I. Brun-Heath,
P. De Mazancourt,
J.-L. Serre and
E. Mornet
Laboratoire SESEP, Université de Versailles-Saint Quentin en Yvelines (A.T., E.M.), F-78035 Versailles, France; Department of Clinical Genetics, Tampere University Hospital (S.-L.S.), Tampere, Finland; and Equipe Structure-Fonction et Génétique, UA 2493, Centre Hospitalier Universitaire Paris Ile de France Ouest, Université de Versailles-Saint Quentin en Yvelines (I.B.-H., P.D.M., J.-L.S., E.M.), Versailles, France
Address all correspondence and requests for reprints to: Dr. E. Mornet, Laboratoire SESEP, Batiment Fermat, Université de Versailles-Saint Quentin en Yvelines, 45 avenue des Etats-Unis, F-78035 Versailles, France. E-mail: etienne.mornet{at}cytogene.uvsq.fr.
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Abstract
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Hypophosphatasia is an inherited disorder due to mutations in the bone alkaline phosphatase (ALPL) gene. We report here a patient with childhood hypophosphatasia diagnosed at 1.4 yr because of pectus excavatum, large anterior fontanel, rachitic skeletal changes, and low serum alkaline phosphatase. Sequencing of the ALPL gene produced evidence of two distinct missense mutations, E174K (c.571G>A), of maternal origin, and a de novo mutation, M45I (c.186G>C). The study of various microsatellite polymorphisms ruled out false paternity and therefore confirmed that M45I occurred de novo in the paternal germline or in the early development of the patient. Site-directed mutagenesis showed that M45I results in the absence of in vitro alkaline phosphatase activity, suggesting that the mutation is a severe allele. In conclusion, childhood hypophosphatasia in this patient is the result of compound heterozygosity for the moderate mutation E174K and a novel severe de novo mutation M45I.
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Introduction
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HYPOPHOSPHATASIA [Mendelian Inheritance in man (MIM) 241500, 241510, and 146300] is an inherited disorder characterized by defective bone and teeth mineralization and deficiency of serum and bone alkaline phosphatase (ALP) activity (1, 2). The bone symptoms are highly variable in their clinical expression, which ranges from stillbirth without mineralized bone to pathological fractures developing only late in adulthood (1, 2). Odontohypophosphatasia is characterized by premature exfoliation of primary teeth with roots intact and/or severe dental caries, not associated with abnormalities of the skeletal system. Severe forms of the disease (perinatal and infantile) are transmitted as an autosomal recessive trait, whereas both autosomal recessive and autosomal dominant transmission may be found in milder forms, especially odontohypophosphatasia (3, 4, 5, 6).
The tissue-nonspecific ALP (TNSALP) gene (ALPL; MIM 171760) is localized on chromosome 1p36.1 (7) and consists of 12 exons distributed over 50 kb (8). More than 160 distinct mutations have been described in the TNSALP gene (for review, see the TNSALP gene mutation database at www.sesep.uvsq.fr/database.html) in North American, Japanese, and European patients, indicating a very strong allelic heterogeneity in the disease (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). This variety of mutations results in highly variable clinical expression and a great number of compound heterozygous genotypes with missense mutations that account for 82% of mutations. The remaining reported mutations are microlesions (11%), splicing mutations (4%), nonsense mutations (3%), and a nucleotide substitution affecting the major transcription initiation site. Until now, de novo mutation has not been reported in this gene, i.e. parental DNA examination showed that the mutations were transmitted. We report here a patient affected with childhood hypophosphatasia due to compound heterozygosity for the missense mutation E174K (c.571G>A) and a novel de novo missense mutation, M45I (c.186G>C).
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Subjects and Methods
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Case report
This patient was studied after informed written consent was obtained from the parents. The patient was diagnosed with hypophosphatasia at the age of 1.4 yr because of pectus excavatum, large anterior fontanel, and rachitic skeletal changes (rachitic rosary) observed in skeletal x-ray examination. Low level of serum ALP (75 U/liter; age-matched normal range, 250-1000 U/liter) together with high urinary phosphoethanolamine (440 µmol/liter; age-matched normal range, 0–78 µmol/liter) confirmed the hypophosphatasia diagnosis. Leukocyte ALP was not detectable, and the serum inorganic phosphorus level was elevated (2.38 mmol/liter; age-matched normal range, 1.30–2.20 mmol/liter). The patient lost most of his deciduous teeth almost immediately after they appeared. He has dental caries in his permanent teeth. During the last 6 yr, the boy has suffered from several pathological fractures in the lower extremities. A first traumatic fracture of left metacarpal occurred at the age of 8 yr. Since then, several fractures affecting both tibia and left femur have occurred. Because of multiple fractures, lower extremities have been surgically treated by internal fixation. During childhood, the patient’s growth declined to –1 SD below average, and at the age of 16.9 yr, his height is 163 cm, his weight is 75.5 kg, and his body mass index is 28.4. No developmental delay has been observed.
The parents of the patient were born in the eastern part of Finland and have no consanguinity. They do not show any clinical symptoms related to hypophosphatasia. Their urinary phosphoethanolamine levels are normal, 85 µmol/liter for the father (age-matched normal range, 82–187 µmol/liter) and 105 µmol/liter for the mother (age-matched normal range, 92–157 µmol/liter), as are their serum ALP levels, 160 U/liter for the father (age-matched normal range, 60–275 U/liter) and 76 U/liter for the mother (age-matched normal range, 50–250 U/liter). The patient also has two older sisters who are healthy and have no symptoms of hypophosphatasia.
Molecular analysis
Sequencing.
Primer sequences of the 12 ALPL gene exons were previously reported (17) and allowed analysis of the whole coding sequence, including intron-exon borders and untranslated exons. PCR products were directly sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase, FS (PerkinElmer, Wellesley, MA) and migrated onto an ABI PRISM 310 electrophoresis system (Applied Biosystems, Foster City, CA). Mutations were confirmed by sequencing two independent PCR products.
Nomenclature of mutations.
The sequence reference used was the cDNA sequence quoted as RefSeq NM_000478.2 in the GenBank database. Nucleotide numbering is given according to the method of Weiss et al. (8) and the recommendations of the Human Genome Variation Society; the first nucleotide (+1) corresponds to A of the ATG initiation codon. Amino acid numbering is given according to Weiss et al. (8) and takes into account a 17-residue signal peptide, i.e. the ATG initiation codon is numbered as residue –17. The letter c indicates nucleotide numbering of the coding sequence, for instance, c.186G>C means that the mutation occurred at position of nucleotide 186 of the coding sequence.
Microsatellite analysis
To identify the four haplotypes present in the family in the ALPL region, two dinucleotide CA repeat polymorphisms located in the region, D1S478 and D1S2725, were analyzed. For each of them, one of the PCR primers was labeled with the 6-carboxyfluorescein ABI dye fluorochrome. PCRs were performed in a final volume of 50 µl with approximately 30 pmol of each primer; 250 µM each of deoxy-ATP, deoxy-CTP, deoxy-GTP, and deoxy-TTP; 1 U Gold Taq DNA polymerase (PerkinElmer); and 1–3 mM MgCl2 depending on the locus amplified. Reactions were heated at 95 C for 10 min and subjected to 30 cycles of 1 min at 95 C, 1 min at the annealing temperature of 50 or 45 C depending on the locus, and 40 sec at 72 C. PCR products were migrated onto an ABI PRISM 310 electrophoresis system (Applied Biosystems).
Paternity testing
Seventeen independent short tandem repeats loci were analyzed by PCR with the SGM Plus and Powerplex16 kits, which were used as recommended by the respective manufacturers [Applied Biosystems and Promega Corp. (Madison, WI)]. Genotyping was performed using the ABI PRISM 310 electrophoresis system. The cumulative paternity index and combined paternity probability were calculated from the allele frequency values. Paternity is considered certain when the paternity index is above 99.99%.
Site-directed mutagenesis and transfection studies
A full-length, wild-type cDNA of the TNSALP gene was obtained by RT-PCR with Pyrococcus furiosis DNA polymerase (Stratagene, La Jolla, CA) and standard molecular biology methods. Mutated cDNA was obtained directly from the normal cDNA using the QuikChange site-directed mutagenesis kit (Stratagene). Mutated and wild-type plasmids were transiently transfected into COS-1 cells for 48 h. Transfections were performed with Lipofectamine Plus reagent (Invitrogen Life Technologies, Grand Island, NY) using the methodology recommended by the manufacturer. The plasmid pcDNA3.1/His/lacZ containing the ß-galactosidase gene was used as a positive control of transfection and expression. ALP and ß-galactosidase activities were determined by monitoring absorbance at 415 and 450 nm, respectively, with commercially available kits. ALP activities were weighted with ß-galactosidase activities.
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Results and Discussion
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Sequencing analysis of exons 4 and 6 of the ALPL gene in the patient revealed the heterozygous mutations M45I (c.186G>C) and E174K (c.571G>A). Sequencing of exons 4 and 6 in the parents showed that the mother was heterozygous for E174K and that the father was homozygous for the normal allele, i.e. he did not carry M45I. The study of various microsatellites indicated that the paternal sample corresponded to the biological father of the patient with an error risk of 10–12 (data not shown), suggesting that the mutation occurred de novo in the paternal germline or in the early development of the patient. Allele distributions of CA repeats D1S478 and D1S2725, both strongly linked to the ALPL gene, and of the intragenic polymorphism S93S (c.330 C>T) revealed four distinct haplotypes in the family (Fig. 1
). The haplotype carrying mutation M45I in the child was inherited from the father, in whom the nucleotide change was absent. An older sister of the patient inherited the same paternal haplotype, but does not have the M45I mutation. The other sister is heterozygous for the maternal mutation E174K and carries the other paternal haplotype. Interestingly, the father’s serum ALP level was in the middle of the normal range, whereas it was borderline in the mother, as frequently observed in heterozygotes. Together, these results confirm that the mutation M45I probably occurred de novo. The mutation M45I was not previously described, and it was of interest to evaluate the effect of this mutation on the protein and its contribution to the phenotype. ALP activity of the mutant enzyme was measured after site-directed mutagenesis and transfection of COS cells. The mean activity of four distinct assays was 0.14% of the wild-type activity (SE, 0.27%). This indicates that the mutation completely abolishes in vitro ALP activity, suggesting that in vivo, its effect is extremely severe. The methionine at position 45 was already present in Escherichia coli, suggesting the important role of this residue (19). Analysis of the three-dimensional model of the enzyme (26) indicates that M45 is very close to some residues of the active site, in particular the histidine at position 362 that participates in zinc coordination is at a distance of less than 4 Å.
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FIG. 1. Segregation of haplotypes linked to the TNSALP gene in the family studied. The index case is shown by an arrow. S93S (c.330C>T) is an intragenic polymorphism, D1S478 and D1S2725 are extragenic CA repeat polymorphisms at distances of 230 and 300 kb from the TNSALP gene, respectively. The alleles are indicated in base pairs corresponding to the size of the PCR products. The nomenclature of mutations is detailed in Subjects and Methods. The index case (shown by an arrow) carries the mutation M45I (c.186G>C), whereas the father who transmitted the haplotype harboring the mutation does not carry the mutation because it occurred de novo.
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The recurrent mutation E174K (c.571G>A), previously described by Henthorn et al. (10), is the most frequent ALPL gene mutation in Caucasians (27) and results from a founder effect occurring in western Europe (28). In contrast with M45I, the mutation has a moderate effect. Indeed, E174K is mostly found in moderate (childhood, adult, and odonto-) hypophosphatasia, and site-directed mutagenesis showed that this mutant protein has a significant residual enzymatic activity (88% of wild-type) (19). The residue E174, not conserved through evolution, is at the surface of the molecule in a region supposed to regulate the approach of the substrate and/or its stabilization; we previously suggested that the polarity of the residue, but not its charge, is essential for substrate steering, explaining the moderate effect of E174K (26). Thus, our patient is compound heterozygous for a severe mutation and a moderate mutation, and its relatively moderate clinical form probably results from expression of the moderate E174K allele. On the basis of the age at diagnosis, the patient is affected with the childhood form of the disease. However, this form is itself very heterogeneous, with some patients affected with very mild bone symptoms and others with severe ones.
The patient reported here is the first reported case of a de novo mutation in the ALPL gene responsible for hypophosphatasia. Although we systematically check the parents for the presence of the mutations found in patients, this was never observed previously. As expected in autosomal recessive diseases, such events are very rare. Because the mutation occurred de novo, the risk of this couple having an affected child in a subsequent pregnancy is very low. However, the existence of germline mosaicism in the father cannot be ruled out, and a molecular prenatal diagnosis may be offered in a subsequent pregnancy, as it is usually offered in diseases due to de novo mutations, such as achondroplasia or Prader-Willi syndrome.
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Footnotes
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First Published Online January 25, 2005
Abbreviations: ALP, Alkaline phosphatase; TNSALP, tissue-nonspecific ALP.
Received July 23, 2004.
Accepted January 17, 2005.
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References
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Abstract
Introduction
