Controlling the Editor: The Many Roles of RNA-Binding Proteins in Regulating A-to-I RNA Editing.

RNA editing is a cellular process used to expand and diversify the RNA transcripts produced from a generally immutable genome. In animals, the most prevalent type of RNA editing is adenosine (A) to inosine (I) deamination catalyzed by the ADAR family. Throughout development, A-to-I editing levels increase while ADAR expression is constant, suggesting cellular mechanisms to regulate A-to-I editing exist. Furthermore, in several disease states, ADAR expression levels are similar to the normal state, but A-to-I editing levels are altered. Therefore, understanding how these enzymes are regulated in normal tissues and misregulated in disease states is of profound importance. This chapter will both discuss how to identify A-to-I editing sites across the transcriptome and explore the mechanisms that regulate ADAR editing activity, with particular focus on the diverse types of RNA-binding proteins implicated in regulating A-to-I editing in vivo.

Methionine synthase reductase deficiency (CblE): A report of two patients and a novel mutation.

IMPORTANCE: Functional methionine synthase reductase deficiency, also known as cobalamin E disorder, is a rare autosomal recessive inherited disease that results in an impaired remethylation of homocysteine to methionine. It presents with macrocytic anemia, hyperhomocysteinemia, and hypomethioninemia, and may also be accompanied with neurological impairment. CLINICAL PRESENTATION: We describe two new cases of unrelated girls with megaloblastic anemia misclassified at first as congenital dyserythropoietic anemia with development of neurologic dysfunction in one of them. INTERVENTION: The posterior finding of biochemical features (hyperhomocysteinemia and hypomethioninemia) focused the diagnosis on the inborn errors of intracellular vitamin B12. Subsequent molecular analysis of the methionine synthase reductase (MTRR) gene revealed compound heterozygosity for a transition c.1361C > T (p.Ser454Leu) and another, not yet described in literature, c.1677-1G > A (p.Glu560fs) in one patient, and a single homozygosis mutation, c.1361C > T (p.Ser545Leu) in the other one. These mutations confirmed the diagnosis of cobalamin E deficiency. CONCLUSION: Treatment with hydroxocobalamin in combination with betaine appears to be useful for hematological improvement and prevention of brain disabilities in CblE-affected patients. Our study widens the clinical, molecular, metabolic, and cytological knowledge of deficiency MTRR enzyme.

Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice.

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.

Macrocytosis, a new predictor for esophageal squamous cell carcinoma in Japanese alcoholic men.

Early esophageal squamous cell carcinoma detected by esophageal iodine staining can be easily treated by endoscopic mucosectomy, and identifying its predictors is important in better selecting candidates to screen for this high-mortality cancer. The common etiologies of elevated mean corpuscular volume (MCV) and esophageal cancer, including folate deficiency, smoking, drinking and high acetaldehyde exposure, suggest testing MCV as such a predictor. Japanese alcoholic men with (n = 65) and without (n = 206) esophageal squamous cell carcinomas, excluding those with liver cirrhosis, were assessed for MCV within 7 days of their last drink, alone or in combination with findings from either the alcohol flushing questionnaire or genotyping to identify inactive aldehyde dehydrogenase-2 (ALDH2*1/2*2) and the less-active form of alcohol dehydrogenase-2 (ADH2*1/2*1), which pose risks for esophageal squamous cell carcinoma. MCV was higher in cancer patients than in the control group. MCV was higher in both groups in those who were heavier smokers, had lower body mass index (BMI), experienced alcohol flushing, and had ALDH2*1/2*2. After adjusting for age, drinking and smoking habits, BMI and ALDH2/ADH2 genotypes, macrocytosis of MCV > or =106 fl was associated with increased risk for esophageal cancer (OR = 2.75). Men with both MCV > or =106 fl and alcohol flushing had an even higher cancer risk (OR = 5.51). The combinations of MCV > or =106 fl with ALDH2*1/2*2 or ADH2*1/2*1 alone, and both ALDH2*1/2*2 and ADH2*1/2*1 (ORs = 11.44, 21.22 and 319.7, respectively) showed consistently higher risk than the corresponding group with MCV <106 fl (ORs = 7.24, 4.71 and 27.01, respectively). In conclusion, MCV measurement, alone or in combination with the markers of alcohol sensitivity, provides a new means of predicting risk for esophageal squamous cell carcinoma in Japanese alcoholic men.

Functional methionine synthase deficiency due to cblG disorder: a report of two patients and a review.

Functional methionine synthase deficiency due to abnormal methylcobalamin metabolism causes megaloblastic anemia, moderate to severe developmental delay, lethargy, and anorexia in association with homocystinuria. Patients with this disorder of cobalamin metabolism can be classified into two separate groups, cblE or cblG, primarily on the basis of complementation analysis with cultured skin fibroblasts. We describe two unrelated boys, ages 3 and 5 years, with the cblG defect in methylcobalamin synthesis. Both children presented with severe developmental delay, lethargy, anorexia, and megaloblastic anemia. The diagnosis of homocystinuria was delayed in each case due to difficulties with detection of small amounts of homocystine in physiologic samples. The clinical course of cblG disease is favorably altered by treatment with intramuscular hydroxycobalamin. Megaloblastosis in the presence of adequate supplies of cobalamin and folate in the blood must alert the clinician to the possibility of functional methionine synthase deficiency and should prompt a careful search for associated biochemical hallmarks, including homocystinuria/emia.

Alcohol-induced macrocytosis and blood pressure.

The effect of alcohol on blood pressure was studied prospectively in consecutive general practice patients with macrocytosis (MCV greater than or equal to 100 fl). The patients were separated into misuser and non-misuser groups on the basis of the Malmö modified Michigan Alcoholism Screening Test. There was no significant difference in the prevalence of antihypertensive medication between the misuser and non-misuser groups. When patients using antihypertensive medication were excluded and the groups were age-adjusted, male misusers (n = 95) compared to control patients (n = 22) had significantly higher diastolic (88 mmHg and 81 mmHg, respectively, P = 0.001) and systolic (146 mmHg and 137 mmHg, respectively, P less than 0.001) blood pressure values. Female misusers (n = 24), as compared to female non-misusers (n = 59) had significantly higher diastolic (83 mmHg and 82 mmHg, respectively, P = 0.04) but not systolic blood pressure values. Thus, alcohol seems to have a pressor effect predominantly among men. As 72% of men with macrocytosis were alcohol misusers and 41% of them either had elevated systolic or diastolic blood pressure, all patients with macrocytosis should be asked about their alcohol consumption and at least the males should have blood pressure measured.

Methylation of DNA in megaloblastic anaemia.

Methylation of cytosine residues in DNA samples, collected before and serially after cobalamin treatment from patients with cobalamin deficiency, was studied using restriction endonucleases Hpa II and Msp I and an epsilon globin gene probe. There was no evidence of hypomethylation in any of the samples. It was concluded that although hypomethylation of metabolites such as choline occurs, that of DNA is preserved in megaloblastic anaemia.

Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice.

The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.

Thymidylate synthesis and utilization via the de novo pathway in normal and megaloblastic human bone marrow cells.

We have measured the thymidylate synthetase activity of intact bone marrow cells using a 3H2O release assay. The mean thymidylate synthetase activity of vitamin B12- or folate-deficient megaloblastic marrow cells was reduced only in severely anaemic patients. There was a correlation between thymidylate synthetase activity and RBC in patients with megaloblastic haemopoiesis. The mean rate of incorporation into DNA of 6-3H deoxyuridine was similar in megaloblastic and normoblastic marrows. The rate of thymidylate synthesis exceeded its incorporation into DNA in all marrows, and the mean ratio between synthesis and incorporation was similar in normoblastic and megaloblastic patients, being independent of both thymidylate synthetase activity and RBC. Thus de novo thymine nucleotides were not utilized more efficiently in megaloblastic marrow cells. These data suggest that impaired thymidylate synthesis may not be the central defect in megaloblastic haemopoiesis, and that there is only a single pool of thymidine triphosphate in human bone marrow cells.

Megaloblastic anemia and immune abnormalities in a patient with methionine synthase deficiency.

We report a case of methionine synthase deficiency associated with cellular immune deficiency discovered in a 14-year-old boy. Principal findings were: developmental delay, recurrent upper and lower respiratory tract infections, megaloblastic anemia, discovered at 3 months of age, unresponsive to cyanocobalamin and poorly responsive to folinic acid. Biochemical studies showed: an abnormal deoxyuridine suppression test despite normal serum folate, cobalamin and transcobalamin levels; a normal intracellular uptake of these two coenzymes; and an absolute requirement of methionine for fibroblast growth, suggestive of defective methionine synthesis. An absence of methionine synthase activity in the patient's bone marrow and a profound depression of this activity in lymphocytes and liver were found. Hypergammaglobulinemia with variable lymphopenia, depressed lymphocyte transformation after lectin or recall-antigen stimulation, defective delayed-type hypersensitivity and decreased natural killer activity were noted as well. The patient died at the age of 14.

Vitamin B12-responsive neonatal megaloblastic anemia and homocystinuria with associated reduced methionine synthase activity.

We present findings on an infant with neonatal megaloblastic anemia, homocystinuria, and neurologic dysfunction that included developmental delay and tonic seizures. There was no methylmalonic aciduria. Cyanocobalamin therapy was accompanied by complete hematologic and neurologic recovery, diminished homocystine excretion, and subsequently normal neurologic development. Cultured fibroblasts and lymphoblasts showed a reduced methionine synthase activity and a growth requirement for methionine. Cobalamin incorporation by the patient's lymphoblasts was normal, but the proportion of cellular methylcobalamin in the patient's lymphoblasts and fibroblasts were markedly reduced and that of adenosylcobalamin normal. The reduced methionine synthase activity was independent of assay reducing (thiol) conditions, but normal levels of activity accompanied culture of the patient's lymphoblasts in medium with markedly increased cobalamin concentration. The characteristics of the reduced methionine synthase of our patient differ significantly from that of the previously described infant with cobalamin E disease and suggest that genetic heterogeneity may characterize this mutation.

Hematopoietic and gastric uracil-DNA glycosylase activity in megaloblastic anemia and in atrophic gastritis with special reference to pernicious anemia.

The activity of the DNA excision repair enzyme uracil-DNA glycosylase was measured in peripheral blood mononuclear cells and in bone marrow aspiration samples obtained from patients with pernicious anemia (PA) or other types of megaloblastic anemia (one case of tapeworm anemia and three cases of myelodysplastic syndromes). In addition, the expression of uracil-DNA glycosylase was investigated in biopsies from the antrum and body of the stomach obtained from nine PA patients, from five patients having atrophic gastritis (AG) not associated with PA, and from six control patients having transient upper abdominal complaints without AG. Our results revealed that there was a considerable interindividual variation in gastric uracil-DNA glycosylase activity. No clear correlation between the enzyme level and the level of gastric atrophy was noted, although AG is generally regarded as a risk factor of gastric cancer. Furthermore, uracil-DNA glycosylase activities in peripheral blood mononuclear cells and in bone marrow cells in PA and in myelodysplastic syndromes were similar to the activities observed previously in non-hematological patients and healthy persons. Transient uracil incorporation into DNA may have a role in the cellular abnormalities associated with megaloblastic hematopoiesis. The present findings demonstrated that the enzymatic activity required for rapid removal of uracil from DNA is also expressed in the megaloblastic state.

A cobalamin metabolic defect with homocystinuria, methylmalonic aciduria and macrocytic anemia.

We have identified a patient with methylmalonic aciduria and homocystinuria due to a defect in cobalamin metabolism of the cb1C type mutant. At the time of admission at eight months of age the patient was malnourished, hypotonic and had macrocytic anemia. Neonatal screening for hypermethioninemia associated with homocystinuria had been normal. Serum vitamin B12 was markedly increased and folate concentration was above normal, as were urinary homocystine and methylmalonic acid. The patient had abnormal brain stem auditory and visual evoked potentials. Fibroblast activity of N5-methyltetrahydrofolate: homocysteine methyltransferase was reduced to approximately 10% of concurrent controls. A course of therapy with hydroxocobalamin resulted in a 90% reduction in excretion of methylmalonic acid and normalization of the evoked potentials. These studies support the efficacy of hydroxocobalamin therapy in this disease, suggest that methylmalonic acid may be the most appropriate metabolite to monitor for therapeutic response, and in importance of electrophysiologic studies in character in objectively monitoring the response to treatment metabolic disease.

Platelet monoamine oxidase activity in megaloblastic anaemia.

Platelet monoamine oxidase activity has been measured in 17 patients with megaloblastic anaemia due to either vitamin B12 or folate deficiency, and in 20 healthy subjects. There was a highly significant increase in patients compared with controls. In two patients, platelet activity decreased following successful treatment. A significant correlation between platelet activity and the severity of bone marrow megaloblastic change, assessed by the deoxyuridine suppression test and bone marrow morphology, was also observed. If the change in activity also occurs in the nervous system, this may contribute to the mental disturbance associated with vitamin B12 or folate deficiency.

[Cytochemical aspects in bone marrow cells in macrocytic anemias]. / Zytochemische Befunde an den Knochenmarkzellen bei makrozytären Anämien.

Cytochemical investigations are useful for the characterization of different kinds of macrocytic anemias. Vitamin B12 and folate defects or chronic alcoholic myelopathy, induce in the erythroblasts cytochemical patterns which can be distinguished from those seen in preleukemia, erythroleukemia, or in drug induced toxic anemia. Tests for alpha-naphthol-acetate-esterase, for acid phosphatase, for iron, and for polysaccharides (PAS-stain), are especially valuable for these diagnostic procedures.

Thymidine kinase in megaloblastic anaemia.

Thymidine kinase has been measured in phytohaemagglutinin (PHA)-stimulated lymphocytes from 13 normal subjects and eight patients with megaloblastic anaemia. The levels in normal subjects ranged from 0.20 to 2.10 units/mg protein (mean 0.903 units/mg protein) and in megaloblastic anaemia from 2.99 to 9.97 units/mg protein). All the patients showed raised levels of the enzyme which were partly but not completely reduced to normal by addition of folic acid in vitro. Vitamin B12 in vitro had a lowering effect in the five vitamin-B12-deficient patients and two patients with combined deficiencies but not in one 'pure' folate-deficient patient. Thymidine kinase activity was highest in the cells of the least anaemic patients, suggesting that the degree of anaemia in megaloblastic anaemia may be determined in part by the ability of the cells to utilize thymidine by the 'salvage' pathway when the de novo pathway of thymidylate synthesis is failing. The rise in thymidine kinase activity in megaloblastic anaemia is presumably due to induction of the enzyme. Addition of methotrexate or 5-fluorouracil, drugs known to inhibit de novo thymidylate synthesis, caused an increase in thymidine kinase activity in normal PHA-stimulated lymphocytes after 24 h (but not after 1 h) which could be completely blocked by addition of puromycin. Thymidine mono- and di-phosphate kinases were also measured in normal PHA-stimulated lymphocytes. The activities were substantially higher than that of thymidine kinase and their activities were unaffected by methotrexate addition.