Hunter disease (mucopolysaccharidosis type II) associated with unbalanced inactivation of the X chromosomes in a karyotypically normal girl.

The mechanism of profound generalized iduronate sulfatase (IDS) deficiency in a developmentally delayed female with clinical Hunter syndrome was studied. Methylation-sensitive RFLP analysis of DNA from peripheral blood lymphocytes from the patient, using MspI/HpaII digestion and probing with M27 beta, showed that the paternal allele was resistant to HpaII digestion (i.e., was methylated) while the maternal allele was digested (i.e., was hypomethylated), indicating marked imbalance of X-chromosome inactivation in peripheral blood lymphocytes of the patient. Similar studies on DNA from maternal lymphocytes showed random X-chromosome inactivation. Among a total of 40 independent maternal fibroblast clones isolated by dilution plating and analyzed for IDS activity, no IDS- clone was found. Somatic cell hybrid clones containing at least one active human X chromosome were produced by fusion of patient fibroblasts with Hprt- hamster fibroblasts (RJK88) and grown in HAT-ouabain medium. Methylation-sensitive RFLP analysis of DNA from the hybrids showed that of the 22 clones that retained the DXS255 locus (M27 beta), all contained the paternal allele in the methylated (active) form. No clone was isolated containing only the maternal X chromosome, and in no case was the maternal allele hypermethylated. We postulate from these studies that the patient has MPS II as a result of a mutation resulting in both the disruption of the IDS locus on her paternal X chromosome and unbalanced inactivation of the nonmutant maternal X chromosome.

Properties of partially purified goat kidney beta-D-mannosidase.

Lysosomal beta-D-mannosidase (EC 3.2.1.25) was purified 6900-fold from normal goat kidney by serial Concanavalin A-Sepharose 4B and Red A dye ligand affinity chromatography, followed by anion exchange and molecular sieve high performance liquid chromatography. The relative molecular mass of the enzyme was estimated by molecular sieving to be 79,000 +/- 3000. The apparent Km for the synthetic substrate, 4-methylumbelliferyl-beta-D-mannopyranoside, was 2.3-2.8 mM and the sharp, unimodal pH optimum was 5.5. Enzyme activity was inhibited by Hg2+, Zn2+, Ag+, Co2+ and the thiol reactive agent N-ethylmaleimide. The mannose derivatives p-nitrophenyl-beta-D- thiomannopyranoside and p-aminophenyl-beta-D-thiomannopyranoside inhibited enzyme activity and may be of use as immobilized ligands in future attempts to purify beta-D-mannosidase by specific affinity chromatography.

Caprine beta-D-mannosidosis: characterization of a model lysosomal storage disorder.

Interest in using caprine beta-D-mannosidosis as a model to evaluate bone marrow transplantation in the treatment of human lysosomal storage disorders provided the stimulus for characterization of beta-D-mannosidase in selected goat tissues and induction of hemopoietic chimerism in the goat. Total beta-D-mannosidase activity was measured with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. Residual activity in mutant liver was 52% of control but no activity was detectable in mutant kidney or brain tissue. Normal adult goat liver contained two forms of beta-D-mannosidase, a nonlysosomal form (52%) with a broad pH range for optimum activity (4.5-8.0) and a lysosomal form (48%) with a pH optimum of 5.5. Residual enzyme in mutant liver consisted entirely of the nonlysosomal form. Normal adult thyroid, kidney and brain contained two major lysosomal isoenzymes with pIs 5.5 and 5.9 and traces of a minor isoenzyme with pI 5.0. Normal liver contained three isoenzymes with similar pIs; however, an isoenzyme with pI 5.0 predominated. In 60-day fetal liver lysosomal isoenzymes predominated and only trace amounts of nonlysosomal isoenzyme were detectable. Total hepatic beta-D-mannosidase activity increased towards adult levels during the last 90 days of gestation as a result of increasing nonlysosomal isoenzyme activity. Intraperitoneal injection of fetal liver cells into 60-day goat fetuses resulted in sustained hemopoietic chimerism in surviving kids without evidence of graft-versus-host-disease. These results suggest that transplantation of normal fetal liver cells into preimmunocompetent goat fetuses affected with beta-D-mannosidosis is feasible and may provide an alternative strategy for evaluation of postnatal bone marrow transplantation in the treatment of human lysosomal storage disorders.

Induction of hemopoietic chimerism in the caprine fetus by intraperitoneal injection of fetal liver cells.

Intraperitoneal injection of allogeneic liver cells from 43-day-old male fetuses into normal 60-day female goat fetuses resulted in persistent hemopoietic chimerism in surviving recipients without clinical evidence of graft-versus-host disease. Transplantation of normal fetal liver cells into preimmunocompetent goat fetuses affected with beta-D-mannosidosis may provide an alternative strategy for evaluating hemopoietic stem cell transplantation in the treatment of human lysosomal storage diseases.

Properties and prenatal ontogeny of beta-D-mannosidase in selected goat tissues.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.