Mutation of a new gene causes a unique form of Hermansky-Pudlak syndrome in a genetic isolate of central Puerto Rico.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous albinism and a storage pool deficiency due to an absence of platelet dense bodies. Lysosomal ceroid lipofuscinosis, pulmonary fibrosis and granulomatous colitis are occasional manifestations of the disease. HPS occurs with a frequency of one in 1800 in north-west Puerto Rico due to a founder effect. Several non-Puerto Rican patients also have mutations in HPS1, which produces a protein of unknown function. Another gene, ADTB3A, causes HPS in the pearl mouse and in two brothers with HPS-2 (refs. 11,12). ADTB3A encodes a coat protein involved in vesicle formation, implicating HPS as a disorder of membrane trafficking. We sought to identify other HPS-causing genes. Using homozygosity mapping on pooled DNA of 6 families from central Puerto Rico, we localized a new HPS susceptibility gene to a 1.6-cM interval on chromosome 3q24. The gene, HPS3, has 17 exons, and a putative 113.7-kD product expected to reveal how new vesicles form in specialized cells. The homozygous, disease-causing mutation is a large deletion and represents the second example of a founder mutation causing HPS on the small island of Puerto Rico. We also present an allele-specific assay for diagnosing individuals heterozygous or homozygous for this mutation.

Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse.

The osteochondrodysplasias are a genetically heterogeneous group of disorders affecting skeletal development, linear growth and the maintenance of cartilage and bone. We have studied a large inbred Pakistani family with a distinct form of recessively inherited spondyloepimetaphyseal dysplasia (SEMD) and mapped a gene associated with this dwarfing condition to chromosome 10q23-24, a region syntenic with the locus for the brachymorphic mutation on mouse chromosome 19. We identified two orthologous genes, ATPSK2 and Atpsk2, encoding novel ATP sulfurylase/APS kinase orthologues in the respective regions of the human and mouse genomes. We characterized a nonsense mutation in ATPSK2 in the SEMD family and a missense mutation in the region of Atpsk2 encoding the APS kinase activity in the brachymorphic mouse. ATP sulfurylase/APS kinase catalyses the metabolic activation of inorganic sulfate to PAPS, the universal donor for post-translational protein sulfation in all cell types. The cartilage-specificity of the human and mouse phenotypes provides further evidence of the critical role of sulfate activation in the maturation of cartilage extracellular matrix molecules and the effect of defects in this process on the architecture of cartilage and skeletogenesis.

Lysosomal elastase and cathepsin G in beige mice. Neutrophils of beige (Chediak-Higashi) mice selectively lack lysosomal elastase and cathepsin G.

A profound decrease in activities of the two lysosomal serine proteinases, elastase, and cathepsin G, was found in neutrophils of four independent beige mutants. Elastase and cathepsin G activities were assayed with the specific synthetic substrates MeO-Suc-Ala-Ala-Pro-Val-MCA and Suc-Ala-Ala-Pro-Phe-pNA, respectively. The defect is intrinsic to cells of beige mice, since transplantation of bone marrow from normal to mutant mice restored normal proteinase activity, and normal mice transplanted with beige marrow produced neutrophils with a deficiency of proteinase activity. The loss of elastase and cathepsin G activity was confirmed by separation of [3H]diisopropylfluorophosphate-labeled proteins on denaturing gels, which also revealed that other serine proteinases are at normal levels in beige neutrophil extracts. The deficiency of lysosomal proteinase activity appears specific, in that four other common neutrophil lysosomal enzymes, plus the spectrum of major neutrophil proteins are not affected by the beige mutation. The deficiency of proteinase activity is likely not the primary genetic alteration of the beige mutation, since more than one proteinase is affected, and heterozygous F1 mice have normal rather than intermediate levels of both proteinases. The lowered proteinase activity may contribute to the high susceptibility of beige mice and Chediak-Higashi patients to infection.

Elastase and cathepsin G activities are present in immature bone marrow neutrophils and absent in late marrow and circulating neutrophils of beige (Chediak-Higashi) mice.

Elicited peritoneal neutrophils of beige (Chediak-Higashi) mice essentially lack activities of the neutral serine proteinases elastase and cathepsin G, which may explain the increased susceptibility to infection of beige mice and Chediak-Higashi patients. We have examined neutrophils of beige mice at earlier points in their development to determine if the proteinase genes are never expressed or whether they are expressed and then lost during neutrophil maturation. Surprisingly, bone marrow of beige mice had significant elastase and cathepsin G activity (approximately 60% of normal). The results of several experiments indicate that neutrophils were the sole source of elastase and cathepsin G in bone marrow. Neutral proteinase activity was readily demonstrable by histochemical procedures in beige marrow neutrophil precursors up to and including the metamyelocyte stage. However, mature neutrophils of beige marrow had greatly decreased activity. Also mature neutrophils (PMNs) of the peripheral circulation, like peritoneal neutrophils, had very low elastase and cathepsin C activities. Thus we conclude that beige neutrophil precursors express neutral proteinase activity, which is largely and irreversibly depleted by the time they fully mature in marrow.

Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice.

The beige mouse is an animal model for the human Chediak-Higashi syndrome, a disease characterized by giant lysosomes in most cell types. In mice, treatment with androgenic hormones causes a 20-50-fold elevation in at least one kidney lysosomal enzyme, beta-glucuronidase. Beige mice treated with androgen had significantly higher kidney beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase (hexosaminidase) levels than normal mice. Other androgen-inducible enzymes and enzyme markers for the cytosol, mitochondria, and peroxisomes were not increased in kidney of beige mice. No significant lysosomal enzyme elevation was observed in five other organs of beige mice with or without androgen treatment, nor in kidneys of beige females not treated with androgen. Histochemical staining for glucuronidase together with subcellular fractionation showed that the higher glucuronidase content of beige mouse kidney is caused by a striking accumulation of giant glucuronidase-containing lysosomes in tubule cells near the corticomedullary boundary. In normal mice lysosomal enzymes are coordinately released into the lumen of the kidney tubules and appreciable amounts of lysosomal enzymes are present in the urine. Levels of urinary lysosomal enzymes are much lower in beige mice than in normal mice. It appears that lysosomes may accumulate in beige mice because of defective exocytosis resulting either from decreased intracellular motility of lysosomes or from their improper fusion with the plasma membrane. A similar defect could account for characteristics of the Chediak-Higashi syndrome.

Effects of ammonia on processing and secretion of precursor and mature lysosomal enzyme from macrophages of normal and pale ear mice: evidence for two distinct pathways.

Lysosomal enzymes have been shown to be synthesized as microsomal precursors, which are processed to mature enzymes located in lysosomes. We examined the effect of ammonium chloride on the intracellular processing and secretion of two lysosomal enzymes, beta-glucuronidase and beta-galactosidase, in mouse macrophages. This lysosomotropic drug caused extensive secretion of both precursor and mature enzyme forms within a few hours, as documented by pulse radiolabeling and molecular weight analysis. The normal intracellular route for processing and secretion of precursor enzyme was altered in treated cells. A small percentage of each precursor was delivered to the lysosomal organelle slowly. Most precursor forms traversed the Golgi apparatus, underwent further processing of carbohydrate moieties, and were then secreted in a manner similar to secretory proteins. The lag time for secretion of newly synthesized beta-galactosidase precursor was notably longer than that for the beta-glucuronidase precursor. The source of the secreted mature enzyme was the lysosomal organelle. Macrophages from the pale ear mutant were markedly deficient in secretion of mature lysosomal enzyme but secreted precursor forms normally. These results suggest that ammonia-treated macrophages contain two distinct intracellular pathways for secretion of lysosomal enzymes and that a specific block in the release of lysosomal contents occurs in the pale ear mutant.

Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Mouse liver beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the beta-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, beta-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway.

Recombinant inbred lines: value in the genetic analysis of biochemical variants.

Analysis of inducibility by androgens and electrophoretic mobility of kidney glucuronidase in progenitor and derived recombinant inbred mouse lines suggests that a single major regulatory gene at or near the glucuronidase structural gene on chromosome 5 determines the rate of enzyme accumulation.

Lysosomal dysfunctions associated with mutations at mouse pigment genes.

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.

The murine misty mutation: phenotypic effects on melanocytes, platelets and brown fat.

Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.

The costs of employee smoking. A computer simulation of hospital nurses.

A computer simulation model of the economic impact of employee smoking on an employer was developed with a population of hospital nurses as the example. Net economic impact was calculated by estimating cumulative costs borne by the employer under various scenarios and comparing them with projected costs estimated from baseline data. The model included the following: baseline number of staff, baseline percentage of smokers, annual employee turnover rate, number of smokers interested in quitting, the cost of a smoking cessation program, expected success rate of a voluntary cessation program, smokers quitting spontaneously without a program, and employer-borne costs related to employee smoking. A variety of scenarios were constructed to generate a range of employer net economic impact figures and resulting percentages of smoking employees. Results showed that the benefits of a cessation program would be eliminated over several years, unless the prevalence of smoking in incoming employees was reduced. The most favorable scenario, combining a hospital cessation program and reduced smoking among new employees, generated cumulative savings, discounted at 5%, of $358,000 to $684,000 over an eight-year period.

Progenitor cell defect correctable by bone marrow transplantation in five independent mouse models of platelet storage pool deficiency.

Two human platelet storage pool deficiencies (SPD), Hermansky-Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited and characterized by hypopigmentation, prolonged bleeding, and normal platelet numbers accompanied by a reduction of platelet dense granules. Seven independent and unique mouse pigment mutations regulated by separate genes have been proposed as animal models for SPD. Mice homozygous for the recessive mutations have diluted pigmentation, prolonged bleeding times, normal platelet concentrations, and reduced numbers of platelet dense granules. Reciprocal bone marrow transplantations were carried out between normal C57Bl/6J mice and five of these mutants, pearl, light ear, pale ear, ruby-eye, and maroon, to test whether the platelet defects are due to platelet progenitor cells or to humoral regulatory factors. Recipient mice were transplanted with marrow after 950-rad whole body irradiation. The prolonged bleeding time and low serotonin concentrations of the five mutants were converted to normal values after transplantation with normal marrow. Normal mice displayed characteristics of platelet SPD when transplanted with mutant marrow. This study demonstrates that in each of five independent mouse models the thrombopathy of SPD is due to a platelet progenitor cell defect correctable by bone marrow transplantation. These findings suggest that in severe cases human SPD may be amenable to treatment by bone marrow transplantation.

Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.

We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.

Differential effect of hypophysectomy on the synthesis of beta-glucuronidase and other androgen-inducible enzymes in mouse kidney.

The levels of several androgen responsive enzymes including beta-glucuronidase, alcohol dehydrogenase, D-amino acid oxidase and arginase, were compared in kidneys of normal and hypophysectomized female mice after treatment with testosterone. While hypophysectomy did not alter the basal level of glucuronidase, the androgen-mediated accumulation of kidney beta-glucuronidase was greatly decreased in hypophysectomized mice. Measurements of the rate of synthesis of glucuronidase showed that after androgen treatment the enzyme was synthesized in kidney of hypophysectomized mice at only 5% the normal rate. Glucuronidase activity in seven other organs was not appreciably affected by treatment with androgens or by hypophysectomy. Unlike the effect of hypophysectomy on kidney glucuronidase, there was no reduction in the accumulation of alcohol dehydrogenase or D-amino acid oxidase in kidney of hypophysectomized mice after androgen treatment. Hypophysectomy caused a large reduction in kidney arginase activity. However, subsequent administration of testosterone restored much of this activity. It is concluded that there are at least two mechanisms by which androgens increase enzyme activity in kidney. The normal increase in activity or rate of synthesis of beta-glucuronidase following androgen administration requires pituitary hormones and/or products of these hormones, while the increase in activity of enzymes like alcohol dehydrogenase and D-amino acid oxidase does not require pituitary hormones.

Functional status after coronary artery bypass grafting and percutaneous transluminal coronary angioplasty.

Two cohorts of consecutive patients of comparable age with similar preprocedure cardiac function who underwent either coronary artery bypass grafting (CABG; n = 106) or percutaneous transluminal coronary angioplasty (PTCA; n = 64) were entered into a prospective comparison study examining functional status and return to work during the first year of recovery. Patients were evaluated using standardized functional status instruments for activities of daily living, work performance, social activity, mental health and quality of social interaction at 1, 6 and 12 months after the procedure. Within the CABG group, statistically significant improvements of functional status on every subscale were noted over the 1-year follow-up. Patients undergoing PTCA demonstrated significant improvement in all dimensions except for the quality of interaction at 1 year as compared with baseline. When the 2 groups were compared, the PTCA group demonstrated greater participation than the CABG group in routine daily physical and social activities at 1 and 6 months, but this apparent advantage disappeared by 1 year. Measures of psychological functioning were better after CABG than after PTCA. A reduction in the number of those with employment occurred in both the CABG and PTCA groups, independent of physical functional status measures, which improved in both groups after the procedures. For those with employment, the CABG group reported the greatest improvement in work performance.

Roles of growth hormone and testosterone in the synthesis of mouse kidney glucuronidase.

Mouse kidney beta-glucuronidase production is under multihormonal control. In normal mice, kidney glucuronidase is induced over 100-fold by testosterone. However, hypophysectomy reduces this induction to about 5% of normal. This loss in inducibility was in part restored by growth hormone. Simultaneous administration to hypophysectomized female mice of growth hormone and testosterone, but not of prolactin and testosterone, restored kidney glucuronidase concentration to half that found in testosterone-treated normal female mice. Growth hormone alone had no effect in hypophysectomized females nor did it enhance glucuronidase activity in testosterone-treated normal females. Radiolabeling experiments demonstrated that the enhancement by growth hormone of glucuronidase activity was accompanied by a corresponding increase in its rate of synthesis. Kidney hypertrophy and kidney glucuronidase production may be under common hormonal regulation. Testosterone or growth hormone treatment alone of hypophysectomized mice had little or no effect on either process, but combined treatment with the two hormones significantly enhanced both. The rate of synthesis of kidney glucuronidase is controlled by the Gur gene. Relative differences in kidney glucuronidase synthesis in mice of different Gur genotype were maintained in testosterone-treated hypophysectomized mice. This suggests that control of glucuronidase synthesis by the Gur locus is exerted by interaction with androgens rather than pituitary products.

von Willebrand's variant (type II Buffalo). Thrombocytopenia after desmopressin but absence of in vitro hypersensitivity to ristocetin.

Von Willebrand's disease is categorized into types and subtypes based on multimeric analysis of plasma von Willebrand's factor. Such categorization is of value because both the mode of inheritance and the choice of therapeutic material differ between subtypes. The Type IIB variant is characterized by hypersensitivity in vitro to ristocetin and thrombocytopenia after administration of desmopressin (DDAVP). Hypersensitivity to ristocetin has also been described in Type I variants but without thrombocytopenia after DDAVP. This report describes a new Type II variant characterized by the converse situation, absence of hypersensitivity to ristocetin in vitro but transient thrombocytopenia after intravenous administration of DDAVP.

Factors related to functional status after coronary artery bypass surgery.

We studied physical functioning and social and leisure functioning of 125 men before, 1 month after, and 6 months after coronary artery bypass surgery. Relationships between functional status outcomes and selected psychosocial and physical variables were examined. Although functional status outcomes improved significantly 6 months after surgery, 13% of patients continued to report important functional status disabilities, and 45% reported difficulty in or no participation in moderately vigorous activities. The variable most predictive of functional status was the patient's estimation a priori of his ability to carry out specific activities (self-efficacy). Other psychosocial and physical factors, other than postoperative treadmill performance, did not add significantly to the prediction of the outcomes. Findings suggest that rehabilitative programs attempting to facilitate physical and social reintegration of the patient after coronary artery bypass surgery should test the effects of interventions to increase self-efficacy.