Search

The clinical presentation of mitochondrial DNA (mtDNA) disorders is quite diverse. Very often, the initial symptoms do not fit a specific disease, and diagnosis is difficult to make. We describe a patient who presented with macrocytic anemia. Extensive biochemical and clinical work-up failed to provide an etiology for the macrocytic anemia. The patient over the course of 6 years developed gait problems, exercise intolerance, episodic vomiting, short stature, dermatological problems, and recurrent infection. At age 8 years she had encephalopathy with ataxia and dysphagia. The presence of elevated lactate, bilateral basal ganglia calcification, and ragged red fibers led to mtDNA mutational analysis. A novel 4.4-kb deletion from nucleotide position 10,560 to nucleotide position 14, 980 was identified in muscle biopsy. The same heteroplasmic mtDNA deletion was present in blood, buccal cells, and hair follicles, but not in mother's blood, consistent with sporadic mutation in the patient. This case emphasizes the importance of considering mtDNA disorder in patients with multisystemic symptoms that cannot be explained by a specific diagnosis.

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.

The DNA of 287 healthy white elderly volunteers in the New Mexico Aging Process Study, between 63 and 91 years of age, was examined for mutations of the HLA-H gene at nt 845 and nt 187. None were found to be homozygous for the 845A mutation and there were no gender differences in the percentage of the various mutations. The frequency of the 845A mutation was 0.061 resulting in a carrier frequency of 12.2%. The frequency of the 187G mutation was 0.136 resulting in a carrier frequency of 19.9% for a single mutation; 2.4% were compound heterozygous, 834A/187G and 2.4% were homozygous for the 187G mutation. After excluding 5 men and 4 women with microcytic or macrocytic anemia, mean percent transferrin saturation (PSAT) and iron stores, as estimated from serum ferritin concentrations, were calculated for each mutation. Estimated iron stores were normally distributed (range approximately 50 to 1,550 mg) with men (n=111) having significantly higher mean estimated iron stores than women (n=167), 826 +/- 318 and 753 +/- 287 mg, respectively. More men, 15 of 28, (54%) with estimated iron stores in the upper quartile, >/= 1,050 mg, had a HLA-H mutation compared to 25 of 83 (30%) who had a mutation and whose estimated iron stores were < ,050 mg, P<0.05. Seven were heterozygous for the 845A mutation with mean estimated iron stores of 1,300 +/- 127 mg, 7 were heterozygous for the 187G mutation with mean estimated iron stores of 1,439 mg. Similar differences were not noted in women. Even though the potential role of the 187G mutation in the phenotypic expression of HH is less certain than the 845A mutation, the increase in PSAT seen in men with the 187G mutation and the equal distribution of 845A and 187G mutations seen with iron stores >/= 1,050 mg lends support for the involvement of the 187G mutation, or a linked mutation, in iron metabolism. We concluded that men having either a single chromosomal 845A and/or 187G mutation results in higher PSAT's and estimated iron stores than if no HLA-H mutation were present.

We describe a pedigree with maternally inherited sideroblastic anaemia in which the red cells are dimorphic with a raised MCV. To our knowledge, this form of hereditary sideroblastic anaemia (HSA) has not been reported previously. 16 members of the family were investigated, revealing eight affected members. Two further family members were not tested but were presumed affected on the histories available. The proband, born in 1967, presented during pregnancy with a macrocytic anaemia (Hb 7.0 g/dl, MCV 106 fl) and a dimorphic red cell picture. Post partum, a bone marrow biopsy showed hypercellularity, mild dyserythropoiesis and ring sideroblasts. Cytogenetics were normal. Other causes of macrocytosis were excluded. Six other family members (three female, three male) have similar findings. There is no evidence of paternal transmission. An additional female relative who presented in 1992 with refractory anaemia with excess blasts in transformation and a dimorphic blood film, died from progression to AML. Affected members show a raised metal-free red cell protoporphyrin level suggestive of a defect at the level of Fe2+ incorporation into protoporphyrin. We propose that this form of HSA is due to a mitochondrial mutation. A search for deletions or point mutations in the mitochondrial DNA is currently underway.

Macrocytosis in the elderly is often caused by abnormalities of haematological stem cell differentiation. In this study, a group of elderly patients was analysed for four molecular and cell biological parameters. The aim of the study was to screen elderly patients with idiopathic macrocytic anaemia or MDS for a set of alterations which are related to haematological dysplasia. The analyses used were: DNA-methylation at the calcitonin A gene 5'-area, NRAS point mutations at codons 12 and 13, in vitro colony formation of peripheral blood progenitor cells and cytogenetics of bone marrow cells. The results show that a significant portion of elderly patients with idiopathic macrocytosis have one or more of the abnormalities analysed. Hypermethylation of the calcitonin A gene 5'-area at the chromosome 11 band p15 is relatively common (7/15). Chromosomal aberrations (3/12) and NRAS oncogene point mutations (0/15) were rare findings. In vitro culture of erythroid progenitor cells was relatively frequently abnormal (7/15). Eight of our nine macrocytic patients who did not fulfill the FAB criteria for MDS had at least one of the alterations studied; this suggests that these patients might represent early phases of a stem cell disorder.

The susceptibility of haemopoietic stem cell deficient W/Wv mice to infection with Plasmodium berghei was examined. The mean survival time of W/Wv mice after the infection was shorter than that of the +/+ mice. Splenomegaly, a characteristic pathological change of the host after infection with malaria parasites was not observed in W/Wv mice. When haemopoietic activity of the infected mice was examined, a substantial increase in number of multipotent haemopoietic stem cells (CFU-S) and the committed stem cells for granulocytes and macrophages (CFU-GM) or for erythrocytes (CFU-E) was observed in the bone marrow and spleen of +/+ but not of W/Wv mice. CFU-S were not detected in W/Wv mice before or after infection. The number of CFU-GM and CFU-E in bone marrow and spleen of W/Wv mice decreased after infection. Bone marrow grafting from +/+ to W/Wv mice 8 weeks before infection prolonged the mean survival time of the mice and effectively restored the number of CFU-S in the spleen of W/Wv mice. These results indicate that multi-potent haemopoietic stem cells play an important role in the host's defence mechanisms against P. berghei-infection.

We have cloned a partial cDNA encoding murine stem cell factor (SCF) and show that the gene is syntenic with the Sl locus on mouse chromosome 10. Using retroviral vectors to immortalize fetal liver stromal cell lines from mice harboring lethal mutations at the Sl locus (Sl/Sl), we have shown that SCF genomic sequences are deleted in these lines. Furthermore, two other mutations at Sl, Sld and Sl12H, are associated with deletions or alterations of SCF genomic sequences. In vivo administration of SCF can reverse the macrocytic anemia and locally repair the mast cell deficiency of Sl/Sld mice. We have also provided biological and physical evidence that SCF is a ligand for the c-kit receptor.

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (Sl/Sld) animals and two control stromal cell lines, one (+/+2.4) from a non-anemic littermate, and one (GBl/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCl density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co-culture of an IL-3-dependent cell line (FDC-P1) with cell-free ECM preparations from an Sl/Sld and a control (GBl/6) stromal cell line, with and without pre-incubation with IL-3. Cell-free ECM preparations from Sl/Sld and control cell lines supported FDC-P1 growth to an approximately equal extent after pre-incubation with IL-3. FDC-P1 growth support by ECM preparations from both cell lines was also observed without IL-3 pre-incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.

The clinical and magnetic resonance imaging findings of a 14-year-old boy with Pearson syndrome are presented. The patient represents the oldest living survivor of the original four patients described by Pearson and associates. This syndrome has recently been found to be associated with an mtDNA deletion. The patient reported here has a deletion similar but not identical to that reported in the literature. Several mitochondrial myopathies have been associated with mtDNA deletions, with considerable overlap between and among the phenotypes and underlying mtDNA deletions. The same may well prove to be true for Pearson syndrome.

Congenital hypoplastic (Diamond-Blackfan) anemia is a rare macrocytic anemia, generally presenting during infancy or childhood. The condition usually occurs sporadically or in a pattern consistent with autosomal recessive inheritance, although autosomal dominant transmission has been proposed in some kindreds. We report the largest known kindred of congenital hypoplastic anemia, with at least 7 affected individuals over 3 generations, and propose that studies of this kindred may be useful for identifying the mechanism by which their genetic abnormality results in congenital hypoplastic anemia. Erythropoietic investigations on relatives show no inhibitors of erythropoiesis in serum, T-lymphocytes, or macrophages. Their erythroid progenitor cells (CFU-E and BFU-E) were generally quantitatively normal, and were capable of rapid proliferation, as judged by cell-cycle shortening. However, their erythroid progenitors displayed a relative insensitivity to recombinant erythropoietin, and produced relatively few normoblasts per erythroid progenitor cell. We propose that these and subsequent studies may be helpful in selecting candidate genes responsible for the molecular defect in this kindred.

We have compared the response of mice heterozygous for the Slj allele-which have a stromal defect resulting in mild macrocytic anemia-and their normal +/+ littermates to manipulations of the erythron to further characterize the effects of the Slj allele on hemopoiesis. CFU-S kinetics in Slj/+ mice following plethorization or induction of anemia did not differ from similarly treated +/+ littermates. Under all circumstances, however, Slj/+ mice had a smaller splenic CFU-S population than similarly treated +/+ mice. Slj/+ and +/+ mice responded similarly to induction of hemolytic anemia with a large rise in 59Fe incorporation in spleen and blood. Hypertransfused Slj/+ mice were shown to have a diminished response to exogenous erythropoietin in spleen and blood as compared with their +/+ littermates. We conclude that the regulation of CFU-S kinetics in mutant Slj/+ mice is not anomalous as has been reported to be the case in Sl/Sld mice. The defective stromal tissue of Slj/+ mice, however, does not support a normal-sized CFU-S population, which leads to decreased influx of progenitor cells into the erythropoietin-responsive cell compartment, causing the diminished response of plethorized Slj/+ mice to exogenous erythropoietin.

A case of erythroid hypoplasia with refractory anemia and an interstitial deletion of the long arm of one chromosome No. 5 is described. This 5q- anomaly has been previously described in cases of refractory anemia with erythroid hypoplasia or hyperplasia, and acute myelogenous leukemia. The relationship between the 5q- chromosome and associated clinical features is discussed together with evidence against a geographical localization.

An analysis of the S1/S1d mouse for ferrokinetics and fate of peripheral red blood cells has shown the cause of its anemia to be dual in nature. While the S1/S1d produces red cells at a slightly greater rate than its normal littermate, its bone marrow and spleen appear to be operating near their maximal capacity and will reduce their output if anemic stress is partly relieved. The cause of the moderately high level of erythropoiesis in the S1/S1d is a mean daily loss of 2.5--3.0% of its total blood volume via the intestinal tract.

Production of tissue mast cells was evaluated in genetically anemic mice of W/Wv genotype and was found to be abnormal. In the skin of adult W/Wv mice the number of mast cells/cm was less than 1% of the number observed in the congeneic +/+ mice. No mast cells were detectable in other tissues of the W/Wv mice. After transplantation of bone marrow cells from +/+ mice the number of mast cells in the skin, stomach, caecum, and mesentery of the W/Wv mice increased to levels similar to those of the +/+ mice. These results show that the W/Wv mouse is a useful tool for the investigations concerning the physiologic roles and the origin of mast cells.

A case of hereditary transcobalamin II deficiency with neurological involvement is described. The patient presented in early infancy with megaloblastic anaemia and was treated with folinic acid from 6 weeks of age. The diagnosis of transcobalamin II deficiency was not made until he was 2 years old when he showed severely retarded intellectual development, ataxia and pyramidal deficit in the limbs. Following treatment with hydroxocobalamin, his condition has slowly improved but he has remained with a severe neurological deficit. The consequences of vitamin B12 deficiency on neurological development in infancy are discussed.

Mice homozygous for mutant genes at the W locus have a marked macrocytic anemia that is fatal in some genotypes. The defect is believed to originate in the developmentally pluripotent hematopoietic stem cell population. Anemia is first grossly manifest on day 13 of gestation, when the liver is the chief hematopoietic organ. The known paucity of blood-forming foci in livers of homozygotes and the limited formation of their erythrocytes suggested that such fetuses-unlike normal ones-might have conditions favorable for in utero seeding of genetically normal hematopoietic tissue. If this were accomplished before day 13, the anemia might essentially be prevented, or at least substantially mitigated, and normalcy soon achieved by cell selection. This proved to be the case. Allogeneic normal fetal liver cells were microinjected into the blood vessels of the fetal placenta on day 11 of gestation. Of eight mutant homozygotes born from segregating matings, six (four W/W, two W(v)/W(v)) were successfully populated with donor cells. Strain-specific hemoglobin markers demonstrated replacement of the erythroid lineage with the normal type, the rate of substitution being more rapid in the W/W (ordinarily more anemic) recipients. Strain-specific isozyme differences revealed that white blood cells were also replaced. Thus, the initial selective pressure, hence the W-mutant phenotypic lesion, must have occurred at the pluripotent stem cell stage. The animals remained immunologically tolerant of the donor cells and no graft-versus-host reaction occurred. The early introduction of hematopoietic cells differing genetically from all the other tissues of the animal provides possibilities for tracing normal hematopoietic lineages in vivo, for analyzing cell and tissue interactions, such as those between lymphocytes and thymus, and for clarifying the etiology of other blood or immune insufficiencies or malignancies.

Bone marrow of normal adult mice was found, after transplacental inoculation, to contain cells still able to seed the livers of early fetuses. The recipients' own hematopoietic stem cells, with a W-mutant defect, were at a selective disadvantage. Progression of donor strain cells to the bone marrow, long-term self-renewal, and differentiation into myeloid and lymphoid derivatives was consistent with the engraftment of totipotent hematopoietic stem cells (THSC) comparable to precursors previously identified (4) in normal fetal liver. More limited stem cells, specific for the myeloid or lymphoid cell lineages, were not detected in adult bone marrow. The bone marrow THSC, however, had a generally lower capacity for self-renewal than did fetal liver THSC. They had also embarked upon irreversible changes in gene expression, including partial histocompatibility restriction. While completely allogeneic fetal liver THSC were readily accepted by fetuses, H-2 incompatibility only occasionally resulted in engraftment of adult bone marrow cells and, in these cases, was often associated with sudden death at 3-5 mo. On the other hand, H-2 compatibility, even with histocompatibility differences at other loci, was sufficient to ensure long-term success as often as with fetal liver THSC.

Graft-versus-host reactions of parental cells in F1 hybrids were studied with two unrelated inbred strains of mice that differed at the mouse histocompatibility locus. W-anemic F1 recipients were compared with lethally irradiated normal F1 recipients. Both sets of recipients were populated by marrow and spleen cell grafts from parental and F1 donors. Most W-anemic F1 recipients were cured by parental and F1 cell grafts (except B6 spleen). Even after 13 to 18 months, they showed little or no effect from GVH reactions. Lethally irradiated normal F1 recipients tolerated parental marrow grafts almost as well, but gave dramatically different results with parental spleen grafts. Seventy-nine of 80 irradiated F1 recipients of parental spleen grafts died within 1 month. Unlike lethally irradiated recipients, W-anemic recipients have substantial numbers of their own cells along with the donor cells in their lymphoid tissues. These F1 lymphocytes may interact with parental lymphocytes in vivo to restrain reactions against F1 allogeneic antigens.

Recognition of a new allele (Wf) at the W locus of the mouse is based on genetic analysis and demonstration of defective hair pigmentation, macrocytic anemia, and defects in pluripotent hemopoietic stem cells. Unlike other alleles of the W series, this one does not result in sterility.

A brother and sister of Pakistani origin suffered from sensorineural deafness, diabetes mellitus and a macrocytic anaemia. Their bone marrows showed megaloblastic erythropoiesis and contained many ringed sideroblasts. Electron microscope studies of the bone marrow revealed (1) iron-laden mitochondria in many erythroblasts, (2) non-specific abnormalities indicative of dyserythropoiesis in some erythroblasts, and (3) evidence of ineffective erythropoiesis. The deoxyuridine suppression test indicated that the megaloblastic changes were not caused by an impairment of the methylation of deoxyuridylate. Studies of nucleic acid synthesis in the bone marrow cells showed that the rate of incorporation of [3H]thymidine into DNA was increased and that the rates of incorporation of [14C]glycine and [14C]adenine into both DNA and RNA were essentially within the normal range. The anaemia did not respond to therapy with hydroxocobalamin, folic acid or pyridoxine but responded to 25 mg thiamine, daily, by mouth. In one of the cases a post-thiamine marrow aspirate showed a considerable improvement in both the megaloblastic and sideroblastic changes.

Hematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/Wv mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst-forming units (BFU-E), the in vivo spleen colony-forming units (CFU-S), and the cells that repopulate the erythroid compartment of stem cell-deficient mice were examined. The progenitors of 7-day bursts are dramatically reduced in W/Wv marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU-S, unlike W/Wv, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU-E is one of the cells most severely affected by expression of the mutant gene.

In a girl 10 years of age with selective vitamin B12 malabsorption associated with proteinuria and residual symptoms of funicular myelosis an extensive study of the intestinal and nephrologic functions was done. Repeated Schilling tests pointed to a malabsorption pattern of vitamin B12. Gastric acid and intrinsic factor secretion as well as gastric morphology were normal. There were no antibodies against intrinsic factor and parietal cells in serum. Ileal mucosa showed on light- and electron-microscopy no pathologic changes. Pancreatic exocrine function as well as pH and calcium concentrations in the lumen of the gut were within the normal range. A general malabsorption syndrome could be excluded. A high selective glomerular proteinuria was found through different methods. Inulin clearance was slightly reduced, PAH clearance, however, markedly so. There was no further evidence for renal tubular dysfunction. Renal biopsy showed a minimal proliferative intercapillary glomerulonephritis (minimal changes). In electron-microscopic studies a fusion of a part of the foot processes of the podocytes was found. No familialhistory of the syndrome could be demonstrated in our patient.

Resorption of petrotic bone in osteopetrotic (mi/mi) mice was brought about by transplantation of bone marrow to X-irradiated recipients. In an attempt to learn more about the donor cell line involved in this process, both normal and defective marrow were used. The consequent repopulation of the lympho-myeloid complex was monitored by isoenzymes of glucose phosphate isomerase. The progress of normal marrow grafts was contrasted with that of a defective marrow (We/Wv). Despite the observation with We/Wv marrow showed reduced ability to form colonies in the spleen of an irradiated recipient, this marrow was as effective as normal marrow in inducing resorption of petrotic bone. The primordial stem cell for the osteoclast (haematopoietic stem cell?) is thus not a CFUS. Chimaeras with resolution of osteopetrosis by We/Wv bone marrow may exhibit erythropoiesis from residual stem cells of the host but leucocytes and platelets from the donor.

A female patient with the 5q-syndrome (refractory macrocytic anemia with deletion of the long arm of No. 5 chromosome) was followed-up for a period of 33 months and developed a carcinoma of pancreas. Until death she showed no signs of leukemic transformation. This association has not been described, and the genetic predisposition of 5q-patients to malignancies is discussed.

Mice carrying two mutant W alleles usually have severe macrocytic anemias which result from deficiencies of hematopoietic stem cells (CFUs) (1). Anemic W39/W39 and W41/W41 homozygotes (2) have deficiencies in the numbers of femoral stem cells which correspond to the severities of their anemias. The non-anemic W44/W44 homozygote (2) has a few stem cells as the W41/W41 mouse. Nevertheless, bone marrow implants from W44/W44 donors cure the anemias of W/Wv recipients while implants from anemic W39/W39 and W41/W41 donors do not. The peripheral hematologic differences between W41/W41 and W44/W44 homozygotes probably arise from qualitative differences intrinsic to their stem cells rather than from extrinsic hematopoietic factors. The hematopoietic environments of all three W homozygotes are relatively normal in that they support normal erythropoiesis when injected with congenic +/+ marrow. Even non-anemic W44/W44 recipients are repopulated with +/+ donor red cells, indicating that W44/W44 stem cells are at a disadvantage when competing with normal counterparts.

A case of spondyloepiphyseal dysplasia tarda was noted in a dwarf (130 cm tall) 18 years old boy associated with congenital megaloblastic anemia and proteinuria. His two sisters and a cousin are also suffering from similar hematologic disorder. One of his brothers, 145 cm tall, is also involved by spondyloepiphyseal dysplasia, but there is no known hematologic abnormalities. Review of family history revealed that two aunts from mother's side were deceased in adulthood following a chronic anemic disease. The findings in this anemia are compatible with Imerslund-Grâsbech syndrome and coexistence of these two rare genetic disorders in a single family has not been reported previously.

The clinical, hematologic and histologic characteristics of six patients with refractory anemia with deletion of the long arm of chromosome No. 5 are described. These patients had a distinct hematologic picture with macrocytic anemia of mild to moderate severity, normal to low leukocyte count and increased platelet count. The long arm of chromosome No. 5 was deleted in the majority of bone marrow metaphases. The main cause of anemia was underproduction with decreased erythroid precursors in the bone marrow and no increase in peripheral blood reticulocytes. Two of five patients responded transiently to the administration of androgens. In vitro evaluation of the bone marrow growth pattern in semisolid agar culture system was performed in three patients and was found to be normal and distinct from that in patients with preleukemia. In a follow up of up to five years, no patient had changed hematologically and in none had leukemia developed. The 5q-syndrome is a distinct hematologic entity and probably more common than hitherto realized. This diagnosis may have therapeutic and prognostic implications.

Early embryo aggregation derived mouse chimeras have proven an extremely valuable tool to study development of disease processes. In this respect chimaeras prove a unique opportunity to study the interaction between cells genetically pre-destined to become diseased and a normal cell population within the same animal. In this situation chimaeras can be studied in view of possible 'correction' of the disease by the provision of a population of normal host cells and/or their products. Various host deficiency states including classic immune deficiency, potentially 'deficient' anemias and tumours have been studied--the subject of the review here.

Since the description in 1974 of the 5q--syndrome, only 29 cases have been reported. Over a 3 1/2 year period cytogenetic culture of bone marrow submitted from 344 patients being investigated for a blood disorder revealed nine patients with anomalies of chromosome 5. In five of these patients (samples arising from 37 patients being investigated for refractory macrocytic or aplastic anaemia) the 5q-syndrome was diagnosed. The clinical and haematological findings of this syndrome are reviewed and attention is drawn to the importance of reviewing megakaryocytic numbers and morphology in refractory macrocytic anaemia if the diagnosis is to be considered. The diagnosis is compatible with prolonged survival and establishing it prevents repeated and unnecessary investigation.

In allophenic (mosaic) mice produced from blastocysts injected with teratocarcinoma stem cells of the OTT 6050 transplant line, an unexpected coat phenotype led to the discovery that the tumor-lineage cells carried the steel gene (Sl(J)/+). Because steel also causes a macrocytic anemia, mosaics comprising both genetically anemic and normal (+/+) cells fortuitously provided a unique opportunity to examine in vivo the etiology of this anemia in light of previous results indicating that the lesion is extrinsic to the erythroid cells. The experiment differs from previous ones, which involved postnatal grafting, in that here hematopoietic stem cells of anemic and normal genotypes coexist throughout all developmental stages, confronted by tissues of the hematopoietic microenvironment that consist partly or solely of genetically normal cells. Therefore, the possibility exists that the anemia might be completely prevented rather than secondarily ameliorated. Moreover, variation in proportion of normal-strain cells in the hematopoietic supporting tissues could serve to "titrate" minimal requirements to promote normal erythropoiesis. Mice with mixed populations of steel- and normal-genotype cells in blood and other tissues were identified by means of independent markers specific for tumor vs. blastocyst strains of origin. The clinical blood picture of these mosaics proved to be indistinguishable from that of normal controls, even when only a small minority of cells in all tissues of one of the animals were genetically normal. Phenotypic blood normalcy was shown, by occurrence of the typical steel anemia among F(1) germ-line progeny of mosaics, not to be due to any change in the capacity of the mutant gene to elicit the anemia. The results from the mosaics thus demonstrate that the primary expression of the steel lesion is indeed in the hematopoietic microenvironment. However, they also reveal that a surprisingly small complement of normal cells there appears to be adequate to prevent this anemia permanently. The hypothesis is advanced that relatively short-range diffusible substances, produced by cells in the microenvironment and required for normal erythropoiesis, may account for the inductive effectiveness of small cell numbers.

A number of patients with megaloblastic anemia and homocystinuria associated with low levels of methylcobalamin synthesis in cultured cells have been recognized. Methionine biosynthesis by intact cells, as determined by incorporation of label from 5-[14C]methyl-tetrahydrofolate into acid-precipitable material, was deficient in cultured skin fibroblasts that were derived from all of these patients. In one group of patients, activity of the methylcobalamin-dependent enzyme, methionine synthase, in cell extracts was within the normal range when the enzyme was assayed under standard conditions. In a second group of patients, methionine synthase activity was decreased under the same assay conditions. Genetic complementation analysis demonstrated the existence of two complementation classes that corresponded to these two groups of patients. The designation cblE has previously been proposed for normal methionine synthase activity. We propose the designation cblG for the mutation in those patients with methylcobalamin deficiency and decreased synthase activity. The results of these studies suggest that the products of at least two loci are required for cobalamin-dependent methionine biosynthesis in mammalian cells.