Loss of function mutations in the ALPL gene (MIM 171760) result in hypophosphatasia (HPP) (MIM 146300, 241500, 241510), an inherited disorder characterized by defective bone and teeth mineralization and deficiency of serum and bone alkaline phosphatase (AP) activity [1–3]. The gene codes for tissue-nonspecific alkaline phosphatase (TNSALP), a homodimeric phosphomonoesterase anchored at its carboxyl terminus to the plasma membrane by a phosphatidylinositol-glycan moiety [4, 5]. Severe forms of the disease (perinatal and infantile) are transmitted as an autosomal recessive trait while both autosomal recessive and dominant transmission can cause milder forms [1, 6–10]. We, along with other groups, have previously showed that ALPL gene mutations could exhibit a dominant negative effect leading to a mild HPP phenotype in heterozygotes [11–14]. This dominant negative effect may be due to the inhibition of the activity of the wild-type (WT) monomer by the mutated monomer in heterodimers [11, 13, 14] or to the sequestration of the WT protein by the mutated one into the Golgi apparatus, preventing it from being transported to the membrane [12]. Carriers of such mutations may not obviously express the disease. In molecular diagnosis, the detection of a single heterozygous mutation in a patient with mild HPP means that a second mutation remains undetected (intronic mutations or mutations in the regulatory sequence) or that the heterozygous mutation has a dominant negative effect. We and others previously showed that the measurement of in vitro AP activity in COS cells transfected by the mutated cDNA of ALPL correlates with the HPP phenotype [15–21], and that the dominant negative effect of mutations may be tested by co-transfecting mutated and WT cDNAs [13, 14, 21]. Such a tool may therefore help to distinguish true heterozygosity from compound heterozygosity with an undetected mutation. Indeed, a dominant negative effect of a mutation may indicate true heterozygosity while the absence of such effect may suggest compound heterozygosity, i.e. the existence of another undetected mutation.

In order to evaluate how often we could have missed a second mutation in patients with mild HPP where only one mutation was identified, we tested by site-directed mutagenesis the possible dominant effect of 35 mutations carried by these patients. In addition, we genotyped 8 exonic and intronic ALPL gene polymorphisms in the patients and in a control group. This approach should reveal the possible existence of a recurrent undetected intronic mutation detectable by linkage disequilibrium (LD) that could be the cause of the detection failure.

The results show that most of the tested mutations exhibit a dominant negative effect that may account for the mild HPP phenotype of the patients, and for some of the patients, a second mutation in LD with a particular haplotype could not be ruled out.

We show here that a large part of mild HPP (i.e. childhood and adult HPP, odontohypophosphatasia, and perinatal benign HPP) is due to heterozygosity for missense mutations with a dominant negative effect, and that the other part is mostly due to compound heterozygosity for mild and severe alleles. The dominant mutations are severe alleles that inhibit the normal monomer when both the normal and the mutated protein form a dimer. The in vitro inhibition level varied from 19% WT (p.D378V) to 40% WT (p.I72T) that we chose as the upper limit for a dominant effect. Mutations with activities slightly above this threshold could have a dominant negative effect, but overlapping standard deviations of experimental results made the distinction with recessive mutations impossible. Among the 13 remaining patients, 5 carried mutations with significant residual in vitro AP activity: p.A176T (30.2%), p.E191K (56%), p.G249V (34.5%), p.I490F (37.1%) and p.R136H (33.4%). Due to their AP activity and to their frequency in mild HPP patients with two mutations (table 2), p.A176T, p.E191K and p.R136H are clearly mild alleles and it is very likely that the patients carry a second allele not detected by our diagnosis procedure. This is not the case for mutations p.G249V and p.I490F. When co-transfected with the WT allele, the mutation p.G249V does not exhibit a dominant negative effect by inhibition. However we previously showed that this mutation has a dominant negative effect by sequestration of the wild type protein in the Golgi apparatus [12] and thus this mutation should be also classified as dominant Similarly to p.G249V, p.I490F is located on the surface of the molecule, and it is therefore possible that the mutation exhibits a similar effect, but this remains to be demonstrated. The eight other patients carry severe alleles without evidence of an in vitro dominant effect. It is therefore possible that these patients harbor a second mutation not detected by sequencing. Due to the mild phenotype of the patient, such a mutation would be a moderate allele, ruling out a large deletion. An intronic mutation or a mutation in the regulatory sequence could account for these patients, in particular a recurrent mutation in LD with haplotype E could affect patients 37, 39 and 41. But the high frequency of E in the group of patients when compared to the control group suggests that E itself, or a mutation in LD, may play a role in the risk for developing mild HPP. We did not demonstrate any effect of the haplotype on ALPL RNA expression. However, E may have an effect on the enzyme activity itself. Interestingly the SNP c.787T>C that substitutes tyrosine for histidine at position 263 was previously shown to affect the catalytic property of TNAP and bone mineral density (BMD) in old Japanese women [30, 31]. In these studies, the less frequent allele in the Japanese population, allele T, increased the Km and decreased the BMD, and should be therefore considered as the "at risk" allele. By contrast, our study shows that allele C, the less frequent allele in European populations, is associated with haplotype E and should be therefore considered "at risk". The contradiction argues in favor of a causal mutation in LD with C in our HPP patients, rather than in favor of the role of c.787T>C polymorphism itself.

The methodology of cotransfection used here may help the molecular biologist to determine if a patient with mild HPP carries one or two mutated alleles. However, looking at alkaline phosphatase activity in just one cell type, and measuring alkaline phosphatase activity alone should be completed by other studies, especially experiments allowing the detection of abnormalities in intracellular processing and migration, and to distinguish them from changes in catalytic activity on the external surface of cells. Also, it is possible that differences in the RNA expression of the defective allele compared to WT would affect in vitro assessment of the mutation. This should be addressed in the future to fully understand phenotype-genotype relationships in hypophosphatasia.

In conclusion we show here that mild HPP can result from either compound heterozygosity for severe and moderate mutations, but also in a large part from heterozygous mutations with a dominant negative effect. A sequence variation in LD with haplotype E could in addition play the role of an aggravating factor resulting in loss of haplo-sufficiency.