TY - JOUR
T1 - First identification of a gene defect for hypophosphatasia
T2 - Evidence that alkaline phosphatase acts in skeletal mineralization
AU - Weiss, Mitchell J.
AU - Cole, David E.C.
AU - Ray, Kunal
AU - Whyte, Michael P.
AU - Laferty, Mary Ann
AU - Mulivor, Richard
AU - Harris, Harry
N1 - Funding Information:
affected hypophosphatasia patients and 12 control individuals for ALP enzyme activity and L/B/K ALP mRNA1*. Consistent with previous reports13, the cell lines derived from hypophosphatasia patients were deficient in ALP enzyme activity relative to controls. However, all of the patient-derived cells produced normal levels of correctly-sized L/B/K ALP mRNA. It seemed likely that in many of these cell lines the hypophosphatasia mutation resided in the L/B/K ALP gene and would be reflected in the corresponding mRNA sequence. The first patient we chose to study in further detail was a boy born to second cousin parents who are part of an extended inbred kindred from a small community in Nova Scotia, Canada. He exhibited abnormalities typical of severe hypophosphatasia including profound skeletal hypomineralization, serum ALP activity of 11 units/liter (normal, 168-406), and urinary phosphoethanolamine excretion of 4.32 umole/mg creatinine (normal, <0.33). He died at age 3 months. Complementary DNA was synthesized from the patient‘s fibroblast mRNA, and a 1676 base pair segment encompassing the entire protein coding region of the L/B/K ALP cDNA was amplified using the polymerase chain reaction (PCR)14. The amplified segment was inserted into a plasmid vector and the sequences from three separate isolates were compared to normal L/B/K ALP cDNA and genomic sequence^^,^^. One mutation was present in all three isolates: a G to A transition at postion 711 of the cDNAI5, hereafter referred to as mutation 711A, that causes the replacement of alanine-162 of the mature enzyme with threonine. To detect this mutation in the affected pedigree, we used the PCR to amplify the corresponding region of genomic DNA from family members and hybridized the products to two allele-specific oligonucleotide probes14. The patient was homozygous for the mutant allele and the parents heterozygous. To verify that the mutation is responsible for the deficient 4321 [ 1021 M. J. WEISS et al. L/B/K ALP activity observed in the proband, we introduced the G to A transition at position 711 into a normal L/B/K ALP cDNA by site-directed mutagenesis14 and tested the ability of the altered cDNA to express ALP activity. NIH 3T3 cells transfected with the normal cDNA expression plasmid produced ALP activity at a level about 200-fold above backround. In contrast, cells transfected with the mutant plasmid failed to express L/B/K ALP enzyme activity, although they produced the corresponding mRNA and immunologic cross-reactivem aterial. Hence, the mutation observed in the patient results in the synthesis of an enzymatically inactive ALP polypeptide and, therefore, caused hypophosphatasia. Mutation 711A produces an alanine to threonine substitution in amino acid 162 of mature L/B/K ALP. This amino acid position lies between two closely spaced components of the ALP active site which have been determined in the E. coli enzyme16 and are conserved in the human ALPs15: a metal binding site and an arginine residue thought to stabilize the transition state during catalysis. It is possible that the mutation disrupts the spatial relationship between these two catalytically important regions of the protein. Alanine-162 of human L/B/K ALP is conserved at analagous positions in E. coli ALP and numerous mammalian ALPS'~~'~, implying some functional importance. However, the corresponding postion is occupied by serine in yeast ALP17. Mutation 711A is not present in the genomic DNA of 34 unrelated individuals with various forms of hypophosphatasia, consistent with genetic heterogeneity for the disease. Examination of the L/B/K ALP gene in different hypophosphatasia patients, using an approach similar to the one above, should define other mutant alleles that produce this disorder. We are currently studying whether other specific mutations give rise to distinct clinical forms of hypophosphatasia. Acknowledgements: This work was supported by National Institutes of Health grant GM 27018, March of Dimes grant 858, and
PY - 1989
Y1 - 1989
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0024809118&partnerID=8YFLogxK
U2 - 10.3109/03008208909050000
DO - 10.3109/03008208909050000
M3 - Article
C2 - 2605956
AN - SCOPUS:0024809118
VL - 21
SP - 99
EP - 106
JO - Connective Tissue Research
JF - Connective Tissue Research
SN - 0300-8207
IS - 1-4
ER -