Mary Saunders

Lactic acidosis in biotin-responsive multiple carboxylase deficiency caused by holocarboxylase synthetase deficiency of early and late onset

Two patients with biotin-responsive multiple carboxylase deficiency, both presenting with predominant lactic acidosis, are reported. One with disease of early neonatal onset had considerable acute neurologic and persistent dermatologic abnormalities. The other, with late juvenile-onset disease, had chronic neurologic abnormalities without dermatologic findings. Early-onset cases generally have been associated with holocarboxylase synthetase deficiency, whereas those of juvenile onset have been characterized as representing defects in intestinal biotin absorption. However, enzyme analyses of fibroblasts from both patients, grown in biotin-deficient medium, revealed markedly diminished activities of pyruvate, propionyl-CoA, and beta-methylcrotonyl-CoA carboxylases, and all three enzymes showed normal activities after growth in biotin-rich medium. Furthermore, lymphoblast enzyme analysis in the patient with disease of early onset had previously revealed a defect in holocarboxylase synthetase, and fibroblast complementation studies showed that both patients belong to the bio complementation group. These findings indicate that considerable clinical heterogeneity exists among patients with holocarboxylase synthetase deficiency, an observation which does not permit differentiation of the biochemical forms of multiple carboxylase deficiency on the basis of age at onset and clinical presentation.

Prenatal Administration of Biotin in Biotin Responsive Multiple Carboxylase Deficiency

Summary: Biotin responsive multiple carboxylase deficiency was suspected in a third trimester conceptus on the basis of enzymatic confirmation in fibroblasts cultured from an earlier sibling who suffered a demise in the immediate neonatal period. Maternal urinary organic acid profile was normal throughout the final 4 wk of pregnancy. Oral administration of biotin, 10 mg/day to the mother resulted in a 100-fold increase in urinary biotin excretion within 7 days. Urinary biotin excretion over the subsequent 2 wk decreased steadily, suggesting either decreased maternal absorption or increased fetal sequestration.After the birth of nonidentical twins, cord blood and urinary organic acid profiles of the infants were normal. However, cord blood biotin concentration was 4 to 7-fold that of normal newborns. Subsequent enzymatic and genetic complementation studies utilizing cultured skin fibroblasts from the infants demonstrated one of them to be affected by the multiple carboxylase defect, although he was clinically and biochemically normal throughout the neonatal period.Thus, prenatal therapy of this inborn enzymatic defect can be safely and effectively accomplished by administration of pharmacologic biotin doses in the last month of pregnancy.Speculation: The affected infants excellent therapeutic response to pharmacologic doses of biotin, administered to the mother, and the absence of adverse effects in the mother and unaffected twin may eliminate the need for prenatal diagnosis in a pregnancy at risk for multiple carboxylase deficiency.

μ Opiate receptor

Interactions of endogenous peptides with μ opiate receptors are likely to be involved in nociceptive responses in drug-free animals. This chapter presents a study in which the mutant mice were born at the expected frequency, grossly normal, and showed no histologic abnormalities in brain or spinal cord. They were indistinguishable under drug-free conditions from controls in several tests of learning, emotionality, and locomotor skills. However, untreated knockout mice displayed shorter latencies on tail flick and hot plate tests for spinal and supraspinal nociceptive responses compared to wild-type mice. Heterozygous mice exhibited shifts downward and to the right in dose–effect relationships for morphine analgesia. These results suggest the μ opiate receptor has an important role in mediating morphine-induced analgesia.

- MT-I and MT-II

Metallothionins are low molecular weight molecules having high cysteine content (30%). The main function of the Metallothionin I and II (MT-I and MT-II) appears to be detoxification of metals and protection of cells from oxidative stress. MT-I and MT-II are constitutively expressed at low levels and are highly inducible in response to metal ions, glucocorticoids, or lipopolysaccharides. MT-I and MT-II are functionally equivalent enzymes that are localized 6 kb apart in the genome. This chapter presents a study in which MT-1 –/– MT-II –/– mice were viable and fertile, implying that metallothionins had no role in development and reproduction. These mice were more sensitive to hepatic poisoning by cadmium. Livers from cadmium-injected mutant mice displayed localized necrosis, congestion and hemorrhaging, and focal areas of cell degeneration. Moreover, cells from these mice were more susceptible to oxidative stress affecting the intracellular redox status of cells. The results demonstrate that the main function of MT-I and MT-II appears to be detoxification of metals and the protection of cells from oxidative stress.

DNA ligase I

DNA ligase I is thought to be central to DNA replication and repair. DNA ligase I is expressed constitutively with its highest expression occurring in the cells and tissues that are dividing and developing the most rapidly. This chapter presents a study in which the DNA ligase –/– mutation was embryonic lethal by E16.5. Embryos developed normally to midterm. Hematopoiesis then failed to make the transition from yolk sac to fetal liver. Acute anaemia developed despite the presence of erythroid-committed progenitors in fetal liver. This study suggests that DNA ligase I is required for normal development, but is not essential for DNA replication.

- β 2 -microglobulin

β 2 -Microglobulin (β2m) is found in relatively high concentration as a soluble protein in serum. The association of β2m with the MHC class I heavy chain is necessary for the class I complex to be transported from the ER to the cell surface. Thus, the lack of β2m protein results in the functional deletion of MHC class I molecules on the cell surface. β2m-deficient mice are born at normal Mendelian frequencies and are grossly normal. The mutant mice do not have any MHC class I molecules on the surfaces of their cells, although they express low amounts of immature heavy chain in the ER. The mice also fail to express functional Fcγ receptor on the intestinal epithelium of neonates.

von Willebrand factor

von Willebrand factor (vWf ) is a carrier of coagulation factor VIII in plasma, where it protects factor VIII against proteolytic degradation. It circulates in plasma and is found in the basement membrane. vWf is stored in granules, α-granules in platelets, and Weibel–Palade bodies in endothelial cells. It is involved in primary hemostasis by mediating platelet adhesion to the subendothelium in the event of vascular injury. This chapter presents a study in which homozygous vWF-deficient mice were viable and fertile with a highly prolonged bleeding time and decreased factor VIII activity. Spontaneous bleeding events occurred in about 10% of neonates. Defective hemostasis was also observed using a vasculitis model, the Shwartzman reaction. The mice showed severe defects in thrombus formation and arteriolar shear rate. The vWf knockout and heterozygous mice provide a model for the von Willebrand disease.

p45 NF-E2

p45 subunit of nuclear factor-erythroid 2 (p45 NF-E2) is p45 NF-E2 is the hematopoietic-specific subunit of the heterodimeric basic leucine zipper (bZIP) transcription factor NF-E2. The expression of p45 NF-E2 is limited to cell lineages of the hematopoietic system. The murine p45 NF-E2 gene is composed of three exons, with the bZIP region required for protein dimerization and DNA binding located in the third exon. p45 NF-E2-null mice may be useful in blastocyst complementation studies to identify other genes required for platelet development. This chapter presents a study in which investigation of the thrombocytopenia in mice showed that it resulted from a late arrest in megakaryocyte cytoplasmic maturation. Numerous polyploid megakaryocytes were found in the hematopoietic tissues but these cells had a large cytoplasm with a paucity of platelet-specific granules, and a failure to demarcate platelet fields. Small, platelet-like particles were found in association with splenic macrophages but it was unclear whether these reflected platelets generate in the normal manner or simply megakaryocyte debris. Thrombopoietin signaling, as measured by megakaryocyte proliferation, was not perturbed in mutanT-cells. No new DNA-binding complexes containing substitutes for p45 NF-E2 could be identified using gel shift assays with nuclear extracts from p45 NF-E2-null cells. The results show that p45 NF-E2 is essential for platelet production but dispensable for erythrocyte development.

p18 NF-E2

p18 subunit of nuclear factor-erythroid 2 (p18 NF-E2) shares extensive homology with members of the small Maf gene family. All small Maf polypeptides can form heterodimers with p45 NF-E2, but binding with p18 NF-E2 increases the affinity of the transcription factor for the NF-E2 DNA-binding site. The p18 NF-E2 subunit distinguishes the binding activity of NF-E2 from that of AP-1, since p 18 NF-E2 specifically recognizes nucleotides not contain in the AP-1 core sequence. This chapter presents a study in which no differences in the levels of either β-major or ζ-globin transcripts were observed. NF-E2-like DNA-binding activity was present in mutant fetal liver extracts, in spite of the absence of p18 NF-E2, suggesting that another Maf protein is able to substitute for the p18 NF-E2 subunit. These findings show P18 NF-E2 is dispensable in vivo and demonstrates the complex interactions between proteins acting on NF-E2-binding sites in hematopoietic cells.

mdr1b P-glycoprotein

mdr1b is primarily found in bone marrow cells and the hematological compartment, kidney, liver, secretory epithelium of pregnant uterus, placental trophoblasts, adrenal gland, and ovary. It causes multidrug resistance in tumor cells by lowering intracellular drug concentrations. This chapter presents a study in which mdr1b –/– mice displayed normal viability, fertility, and lifespan under normal animal facility conditions. Extensive histological and hematological analyses and analysis of serum clinical chemistry revealed no substantial differences between mutant mice and wild-type mice. No physiological abnormalities or differences in lifespan were observed. The size of the adrenal gland was not altered in mdr1b –/– mice. The mdr1b –/– mice were somewhat hypersensitive to anthracyclines like daunorubicin and doxorubicin. Partially purified hematopoietic progenitor cells of mdr1b –/– mice decreased capacity to extrude the P-glycoprotein substrate and diagnostic dye rhodamine 123. Analysis of the disposition of the cardiac glycoside digoxin showed little effect of the mdr1b knockout on the tissue distribution and elimination of this drug, although the adrenal gland and ovaries accumulate somewhat more digoxin.