Effect of the Pl(A2) alloantigen on the function of beta(3)-integrins in platelets.

The polymorphism responsible for the Pl(A2) alloantigen on the beta(3)-component of beta(3)-containing integrins is reported to be a risk factor for coronary thrombosis. This study examined the effect of Pl(A2) on the function of beta(3)-integrins using platelets from subjects homozygous and heterozygous for Pl(A1) and Pl(A2). There was overlap in the distribution of the dissociation constant (K(d)) and maximum fibrinogen binding (B(max)) values for fibrinogen binding to alpha(IIb)beta(3) on platelets from Pl(A1) and Pl(A2) homozygotes and Pl(A1)/Pl(A2) heterozygotes. However, whereas there was no statistical difference in these values for the Pl(A1) homozygotes and Pl(A2) heterozygotes, the K(d) for the Pl(A2) homozygotes was significantly lower than that for the Pl(A1)/Pl(A2) heterozygotes, but was not statistically different from that for the Pl(A1) homozygotes. No differences were detected in ADP sensitivity between platelets from Pl(A1) homozygotes and Pl(A1)/Pl(A2) heterozygotes, in the IC(50) for RGDS inhibition of fibrinogen binding to alpha(IIb)beta(3), in the alpha(v)beta(3)-mediated adhesion of platelets to osteopontin and vitronectin, and in the phorbol ester-stimulated adhesion to fibrinogen of B lymphocytes expressing alpha(IIb)beta(3) containing either the Pl(A1) or the Pl(A2) polymorphism. Finally, no differential effects of Pl(A2) on turbidometric platelet aggregation, platelet secretion, or platelet thrombus formation were found as measured in the PFA-100. Because no differences were detected in the ability of beta(3)-integrins to interact with ligands based on the presence or absence of the Pl(A2) polymorphism, the results suggest that factors unrelated to beta(3)-integrin function may account for the reported association of the Pl(A2) allele with coronary thrombosis.

Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping.

OBJECTIVE: Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disorder resulting in target organ resistance to the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In many cases, this disorder has been shown to be due to mutations in the gene encoding vitamin D receptors (VDR). In a patient with characteristic features of this disorder, we investigated the functional defect and sequenced the coding region of the gene for mutations. DESIGN: Skin fibroblasts from patient and control were used to measure binding of 1,25(OH)2D3 and functional responses to the hormone. These cells were also used to prepare RNA from which cDNA was prepared and sequenced. Furthermore, genomic DNA was prepared from the fibroblasts and the intron/exon boundaries sequenced. PATIENT: A child with classic features of HVDRR with alopecia diagnosed as having rickets due to resistance to 1,25(OH)2D3. MEASUREMENTS: Nuclear association of 1,25(OH)2D3 was determined in patient and control cells and the functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamin D-24-hydroxylase(24-hydroxylase) activity. VDR cDNA and genomic DNA prepared from patient and control cells were sequenced. RESULTS: Cells from the patient with HVDRR had undetectable amounts of VDR compared to control cells and did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)2D3. Sequencing of the VDR coding region after RT-PCR of RNA revealed an absence of exon 4 in patient RNA which was not due to a deletion in genomic DNA but was caused by exon skipping during RNA processing. In addition, the deletion of exon 4 sequences from RNA leads to a frameshift in translation resulting in a premature stop codon. Amplification of genomic DNA around the intron/exon boundary of exon 4 revealed a point mutation in the 5' donor splice site of intron 4. CONCLUSION: In this study, we have identified a novel mutation in the gene for vitamin D receptors in a patient with the characteristic phenotype of hereditary vitamin D resistant rickets. The mutation at the +5 position in intron 4 is most likely to cause skipping of exon 4 in this patient.

Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures.

OBJECTIVE: Hereditary vitamin D resistant rickets (HVDRR) has been shown to be due to mutations in the gene encoding the vitamin D receptor (VDR). In two patients with the characteristic phenotype we have investigated the functional defect and sequenced the VDR cDNA. We report two new mutations in the DNA binding domain of the VDR gene and we have used the crystallographic structure of the glucocorticoid and oestrogen receptors (GR and ER respectively) as models to explain the stereochemical consequences of these mutations. DESIGN: Patient and control cell lines prepared from skin fibroblasts were used to measure binding of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and functional responses to this hormone. These cells were also used to isolate VDR mRNA from which cDNA was prepared and sequenced. VDR cDNA from affected and control patients was also transfected into receptor defective cells to analyse further functional responses to 1,25(OH)2D3. Computer analysis of mutations in the VDR gene was carried out using the glucocorticoid and oestrogen receptors as model systems. PATIENTS: Two patients with HVDRR from unrelated families. MEASUREMENTS: Cytosolic binding and nuclear association of 1,25(OH)2D3 were determined in control and affected patients, and functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamin D-24-hydroxylase activity (24-hydroxylase). VDR cDNA was sequenced and transfected into VDR-deficient CV-1 cells for further analysis of functional response to 1,25(OH)2D3 following cotransfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS: Cells from HVDRR patients I and II showed detectable numbers of VDR with normal hormone binding. However, unlike controls, the HVDRR cells did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)2D3. Sequencing of cDNA revealed single mutations, in patient I (Phe44-->IIe) and in patient II (Lys42-->Glu). Both these residues are conserved in the steroid/thyroid hormone receptor superfamily and stereochemical analysis has been used to deduce the importance of these amino acids and the deleterious effect of these and other mutations in the DNA-binding domain of the VDR. CONCLUSIONS: Two new mutations in the vitamin D receptor which cause hereditary vitamin D resistant rickets have been described and using molecular modelling we have been able to analyse the genesis of this inherited disease at the level of stereochemistry.

Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene.

OBJECTIVE: Hereditary vitamin D resistant rickets (HVDRR) is characterized by severe rickets and is often accompanied by alopecia. Mutations in the gene encoding the vitamin D receptor have been found in this condition. In a patient with the characteristic phenotype we have investigated the functional defect and sequenced the gene to seek a mutation. DESIGN: Patient and control cell lines prepared from skin fibroblasts and peripheral blood lymphocytes were used to measure binding of 1,25(OH)2D3 and to isolate vitamin D receptor mRNA. VDR cDNA was sequenced and transfected into receptor defective cells. PATIENT: A child with alopecia diagnosed as having rickets due to resistance to 1,25(OH)2D3. MEASUREMENTS: Cytosolic binding and nuclear association of 1,25(OH)2D3 were determined in patient and control cells, and functional response to 1,25(OH)2D3 assessed by measurement of 24-hydroxylase activity. VDR mRNA was prepared, reverse transcribed, and cDNA sequenced. VDR cDNA was also transfected into VDR-deficient CV-1 cells and functional response to 1,25(OH)2D3 assessed by co-transfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS: VDR from the patient were able to bind 1,25(OH)2D3 but showed no nuclear localization resulting in an absence of functional response to 1,25(OH)2D3. Sequencing revealed that the VDR coding region was normal. Expression studies of the patient's VDR showed functionally normal VDR as evidenced by normal transactivation in the presence of 1,25(OH)2D3. CONCLUSION: These data indicate a new cause of tissue resistance to 1,25(OH)2D3 which occurs in the absence of mutations in the coding region of VDR gene and which is characterized by defective nuclear localization of this receptor.

Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3.

We have identified and characterized two mutations in the hormone binding domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a premature stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC) resulting in the substitution of arginine 271 by leucine (R271L) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cells showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, however, be elicited by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the wild-type receptor. This shows that Arg 271 directly affects the affinity of the VDR for its ligand and its conversion to leucine decreases its affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located immediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270) that is conserved among members of the steroid/thyroid/retinoid hormone receptor superfamily. These results represent the first missense mutation identified in the hormone binding domain of VDR and further define the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor.