FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors.

The transcription factors that control lineage specification of haematopoietic stem cells (HSCs) have been well described for the myeloid and lymphoid lineages, whereas transcriptional control of erythroid (E) and megakaryocytic (Mk) fate is less understood. We here use conditional removal of the GATA-1 and FOG-1 transcription factors to identify FOG-1 as required for the formation of all committed Mk- and E-lineage progenitors, whereas GATA-1 was observed to be specifically required for E-lineage commitment. FOG-1-deficient HSCs and preMegEs, the latter normally bipotent for the Mk and E lineages, underwent myeloid transcriptional reprogramming, and formed myeloid, but not erythroid and megakaryocytic cells in vitro. These results identify FOG-1 and GATA-1 as required for formation of bipotent Mk/E progenitors and their E-lineage commitment, respectively, and show that FOG-1 mediates transcriptional Mk/E programming of HSCs as well as their subsequent Mk/E-lineage commitment. Finally, C/EBPs and FOG-1 exhibited transcriptional cross-regulation in early myelo-erythroid progenitors making their functional antagonism a potential mechanism for separation of the myeloid and Mk/E lineages.

Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Homozygous N396T mutation in Gaucher disease: Portuguese sisters with markedly different phenotypes.

Gaucher disease (GD) is characterized by reduced activity of glucocerebrosidase leading to complications in the reticuloendothelial system. N396T, a rarer mutation of the glucocerebrosidase gene, has been encountered in Portuguese populations and has generally been associated with milder phenotypes. This report presents brief histories of two Portuguese sisters, both with homozygous N396T mutations. These patients are phenotypically very different despite the fact that in both patients residual enzyme activity is very low. The case of patient 1 is complicated by comorbid diabetes mellitus and human immunodeficiency virus (HIV) infection. Enzyme replacement therapy (ERT) improved this patient's clinical picture sufficiently to enable antiretroviral treatment to proceed for the HIV. This report demonstrates the poor correlation of clinical GD with genotype as well as with residual enzyme activity. It further illustrates how treatment of the underlying GD with ERT improved symptoms allowing for antiretroviral therapy thereby improving both the GD and HIV.

Gaucher disease among Chinese patients: review on genotype/phenotype correlation from 29 patients and identification of novel and rare alleles.

Gaucher disease, the most prevalent lysosomal storage disease, results from an inherited deficiency in the enzyme glucocerebrosidase. Three clinical forms of Gaucher disease have been described: Type 1 non-neuronopathic, Type 2 acute neuronopathic, and Type 3 subacute neuronopathic. Although Gaucher disease is panethnic, its presentation reveals some ethnic-specific characteristics. The Type 1 form is most common among Caucasian patients. In contrast, the majority of Chinese Gaucher disease patients have early age of onset, severe hematological and skeletal complications, and often neurological involvement, resulting in early childhood death. In this report, we review 29 cases of Gaucher disease from 23 unrelated patients and 6 patients from 3 non-consanguineous families. Among these patients, 13 were diagnosed as Type 1, 10 as Type 2, and 6 as Type 3. A novel mutation, del 205-209ACCTT, was identified in the heterozygous form with mutation R353W (c.1174C>T) by DNA sequence analysis in 2 Type 1 patients who are sibs. Mutation R353W was also found in the heterozygous form in 3 other Type 1 patients, with mutation L444P in 2 sibs and a second unknown Gaucher allele in the third patient. The Gaucher genotypes of the remaining Type 1 patients were F37V/L444P, G46E/L444P, R48W/R120W, N188S/L444P, Y205C/L444P, N370S/L444P, and L444P/unknown. It was noted that mutation N370S in the patient was linked to the pv1.1(-)(1) haplotype present in Jewish patients. Among the Type 2 patients, L444P was present in the heterozygous form with mutation F213I, L385P, or the complex allele (RecNci) in 5 patients. The second most common mutation, F213I, was found in the heterozygous form in 6 patients with mutations N382K, L383R, or L444P. The other mutations found in the Type 2 patients were P122L, V375L, Y363C, M416V, and 383-400del. The genotypes of the 6 Type 3 patients identified were D409H/D409H, D409H/G202R, G46E/N188S, N188S/unknown, and L444P/L444P. While D409H has been reported as being associated with cardiovascular/ocular involvements in Gaucher disease, there have been no such complications in these patients. As noted, the majority of the Gaucher mutations we identified in the Chinese patients were either rare or absent in other populations. With the exception of N370S and R353W found only in the Type 1 form, the majority of these mutations are severe ones that result in poor prognosis and often Types 2 and 3 Gaucher disease.