Cloning and characterization of Hepp, a novel gene expressed preferentially in hematopoietic progenitors and mature blood cells.

Through differential screening of mouse hematopoietic stem cell (HSC) and progenitor-subtracted cDNA libraries we have identified a progenitor cell-specific transcript that represents a novel gene, named Hepp (hematopoietic progenitor protein). The mouse Hepp gene encodes a protein of 237 amino acids with no detectable known functional domains or motifs. Lack of invertebrate orthologs and a high degree of evolutionary conservation of the peptide sequence in vertebrate species (zebrafish, mouse, human) suggest that the Hepp gene could have conserved although as yet unknown function in vertebrates. Mouse Hepp shows a restricted expression pattern in adult tissues and is transcribed at a very low level in heart, lung, spleen, and thymus and at a higher level in muscle. During embryonic hematopoiesis Hepp is not expressed in mouse fetal liver HSC (Sca-1(+)c-kit(+)AA4.1(+)Lin(-) cells), but is abundantly transcribed in the population of hematopoietic progenitors (AA4.1(-) cells). Similarly, during adult hematopoiesis Hepp is not transcribed in the highly enriched population of bone marrow HSC (Rh-123(low)Sca-1(+)c-kit(+)Lin(-) cells), but its expression is upregulated as a greater heterogeneous population of bone marrow HSC (Lin(-)Sca-1(+) cells) differentiates into progenitors (Lin(-)Sca-1(-) cells) and more mature lymphoid and myeloid cell types. A restricted pattern of expression in adult tissues and preferential expression in both fetal and adult hematopoietic progenitors and mature blood cells suggest that Hepp could be involved in molecular regulation of HSC and progenitor cell lineage commitment and differentiation.

FLRF, a novel evolutionarily conserved RING finger gene, is differentially expressed in mouse fetal and adult hematopoietic stem cells and progenitors.

Through differential screening of mouse hematopoietic stem cell (HSC) and progenitor subtracted cDNA libraries we have identified a HSC-specific transcript that represents a novel RING finger gene, named FLRF (fetal liver ring finger). FLRF represent a novel evolutionarily highly conserved RING finger gene, present in Drosophila, zebrafish, Xenopus, mouse, and humans. Full-length cDNA clones for mouse and human gene encode an identical protein of 317 amino acids with a C3HC4 RING finger domain at the amino terminus. During embryonic hematopoiesis FLRF is abundantly transcribed in mouse fetal liver HSC (Sca-1+c-kit+AA4.1+Lin- cells), but is not expressed in progenitors (AA4.1-). In adult mice FLRF is not transcribed in a highly enriched population of bone marrow HSC (Rh-123lowSca-1+c-kit+Lin- cells). Its expression is upregulated in a more heterogeneous population of bone marrow HSC (Lin-Sca-1+ cells), downregulated as they differentiate into progenitors (Lin-Sca-1- cells), and upregulated as progenitors differentiate into mature lymphoid and myeloid cell types. The human FLRF gene that spans a region of at least 12 kb and consists of eight exons was localized to chromosome 12q13, a region with frequent chromosome aberrations associated with multiple cases of acute myeloid leukemia and non-Hodgkin's lymphoma. The analysis of the genomic sequence upstream of the first exon in the mouse and human FLRF gene has revealed that both putative promoters contain multiple putative binding sites for several hematopoietic (GATA-1, GATA-2, GATA-3, Ikaros, SCL/Tal-1, AML1, MZF-1, and Lmo2) and other transcription factors, suggesting that mouse and human FLRF expression could be regulated in a developmental and cell-specific manner during hematopoiesis. Evolutionary conservation and differential expression in fetal and adult HSC and progenitors suggest that the FLRF gene could play an important role in HSC/progenitor cell lineage commitment and differentiation and could be involved in the etiology of hematological malignancies.

Identification and cloning of differentially expressed genes by long-distance differential display.

Differential mRNA display (DD-PCR) amplifies short cDNAs (average size 100-350 bp), representing mainly the 3' untranslated regions (3' UTR) of transcripts. Sequencing of these cDNAs is predominantly uninformative for prediction of function and selection of clones for further analysis. Differential display of longer amplicons (0.5-2.0 kb) could enable isolation of cDNAs that encompass both 3' UTR and at least part of the 3' end of the coding region. The coding sequence information could facilitate selection of candidate clones for further analysis without the necessity of screening cDNA libraries. By combining DD-PCR protocols with long-distance PCR and using hot-start PCR with rTth DNA polymerase we have successfully amplified and comparatively displayed cDNAs ranging in size from 150 bp to 2 kb. Long-distance DD-PCR (LDD-PCR) has generated highly reproducible primer-specific patterns of cDNA fragments, as well as reproducible duplicate fingerprints, obtained from different RNA and cDNA samples. Sequencing and expression analyses of LDD-PCR clones have shown that LDD-PCR (a) enables nonredundant clone sampling, (b) generates many clones that encompass part of the coding region, and (c) samples both abundant and rare transcripts, approximately 60% of which are differentially expressed as confirmed by Northern analysis. Coupled with high-throughput cDNA sequencing and multiplex hybridization of cDNA microarrays for confirmation of differential expression, LDD-PCR could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.

Mild phenotypic effects of a de novo deletion Xpter-->Xp22.3 and duplication 3pter-->3p23.

We report on a girl with a de novo monosomy Xpter-->Xp22.3 and trisomy 3pter-->3p23, normal development and stature, mildly affected phenotype, and learning disabilities with a low normal level of intelligence. Late replication studies using BudR demonstrated that the entire der(X) was inactive in 30% of cells. In 62% of cells the inactivation did not spread to the autosomal segment in the der(X). The normal X was inactivated in 8% of cells. Quantitative X-inactivation studies using the human androgen receptor locus assay (HAR) on peripheral leukocytes and buccal epithelial cells showed extreme skewing of methylation (90.4% of the paternal allele). The correlation of cytogenetic and molecular data suggest that the mild phenotype of the proposita is most likely due to preferential inactivation of the entire der(X), which seems to be of paternal origin.

Application of carrier testing to genetic counseling for X-linked agammaglobulinemia.

Bruton X-linked agammaglobulinemia (XLA) is a phenotypically recessive genetic disorder of B lymphocyte development. Female carriers of XLA, although asymptomatic, have a characteristic B cell lineage-specific skewing of the pattern of X inactivation. Skewing apparently results from defective growth and maturation of B cell precursors bearing a mutant active X chromosome. In this study, carrier status was tested in 58 women from 22 families referred with a history of agammaglobulinemia. Primary carrier analysis to examine patterns of X inactivation in CD19+ peripheral blood cells (B lymphocytes) was conducted using quantitative PCR at the androgen-receptor locus. Obligate carriers of XLA demonstrated > 95% skewing of X inactivation in peripheral blood CD19+ cells but not in CD19- cells. Carrier status for mothers of isolated affected males could be assessed in 10 of 11 families: 7 women showed skewing, and 3 did not. Five carriers were found in six families in which there were no living affected males. Among all those tested, one individual's carrier status was considered to be indeterminate and five women were noninformative for the carrier test. Results obtained by the carrier test were congruent with linkage analysis (where applicable) using the RFLPs DXS178 and DXS94 and two newly developed polymorphic microsatellite markers, DXS178CA and DXS101AAT. Refinements in techniques for primary carrier testing and genetic mapping of XLA now make possible an ordered approach to diagnosis, prenatal diagnosis, and genetic counseling.

Methods for repetitive measurements of multiple hematological parameters in individual rats.

Methods have been developed which permit frequent repetitive blood sampling of rats without perturbing physiological parameters of interest. These techniques allow a comprehensive hematological study over several weeks, in individual rats, thus permitting full documentation of selected parameters during growth and development.