PubMed2002
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.