Abstract 806 Poster Session IV, Tuesday, 5/4 (poster 204)

The physiological functions of several extracellular proteins depend on the modification of particular residues by specific protein hydroxylases. An enzyme defect, or a deficiency in its substrates, can cause clinical symptoms, illustrated by the hydroxylysine-containing collagens. Mutations may affect their structural genes or, as evidenced by Ehlers-Danlos Syndrome VI (EDS VI), the collagen hydroxylase that forms hydroxylysine. Expression of this enzyme varies in the same tissue from different EDS VI patients, and in different tissues from the same patient (Hanauske-Abel et al., FEBS Lett. 110, 73-6; 1980). We postulate that the hydroxyaspartate / hydroxyasparagine-containing fibrillins might be affected similarly not only by mutations in their structural genes, but also by a deficiency of the fibrillin-hydroxylating enzyme, aspartyl 3-hydroxylase (A3H). Such a presumed A3H defect impacts the fibrillin-like LTBPs, which chaperone the major cytokines controlling human bone formation, the TGF-βs (Pfeilschifter et al., J Bone Miner.Res., 716-30; 1998). One consequence of this could be decreased bone density (BD). Using recombinant methods, we have analyzed the molecular architecture of human A3H produced in insect cells. Based on this information, we designed primers for the RT-PCR - based assessment of A3H expression in nucleated cells from the peripheral blood (NCPB) of 7 patients with Marfan syndrome (6 females, 1 male; age 11.2 to 57 years). 6 patients had scoliosis; BD measurements, available on three of them, showed LunarZ scores of -0.9, -2.3, and -3.9. Surprisingly, recombinant human A3H retained its in vitro activity even after deletion of the entire N-terminal half of the native protein. Primers were therefore designed to span nucleotides 1799 to 2101 of the template (Accession U03109), which encode residues indispensible for the C-terminally located catalytic site of A3H. After reverse transcription of RNA isolated from freshly obtained NCPB, radiolabeled PCR products were electrophoretically separated, autoradiographed, and the band intensities calculated relative to the expression of the 'house-keeping' gene β-actin (β-act). In all Marfan parients, the A3H primers generated a signal of the predicted size, identical to the one amplified from authentic human A3H cDNA. The A3H / β-act ratio varied among patients by a factor of 3. However, an association with major symptoms (aortic dilatation, mitral valve prolapse, ectopia lentis, myopia) was not apparent. The mean ratio in pats. with scoliosis, and in those with decreased BD, was in the range of the non-scoliotic, non-osteopenic patient. Our pilot study suggests that NCPB-based A3H expression analysis is possible and could serve as an adjunct to the analysis of the A3H gene in Marfan patients, a step we have initiated.

Supported by NIH RR06020