ABSTRACT
The RCS rat presents an autosomal recessive retinal pigment epithelium dystrophy characterized by the outer segments of photoreceptors being phagocytosis-deficient. A systematic genetic study allowed us to restrict the interval containing the rdy locus to that between the markers D3Mit13 and D3Rat256. We report the chromosomal localization of the rat c-mer gene in the cytogenetic bands 3q35-36, based on genetic analysis and radiation hybrid mapping. Using a systematic biocomputing analysis, we identified two strong related candidate genes encoding protein tyrosine kinase receptors of the AXL subfamily. The comparison of their expression patterns in human and mice tissues suggested that the c-mer gene was the best gene to screen for mutations. RCS rdy- and RCS rdy+ cDNAs were sequenced. The RCS rdy- cDNAs carried a significant deletion in the 5' part of the coding sequence of the c-mer gene resulting in a shortened aberrant transcript encoding a 20 amino acid peptide. The c-mer gene contains characteristic motifs of neural cell adhesion. A ligand of the c-mer receptor, Gas6, exhibits antiapoptotic properties.
Subject(s)
Homozygote , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases , Retinal Diseases/genetics , Sequence Deletion/genetics , Amino Acid Sequence , Animals , Apoptosis/genetics , Base Sequence , Cell Adhesion/genetics , Chromosome Mapping , Crosses, Genetic , Electroretinography , Fluorescein Angiography , Genes, Recessive , Genotype , Inbreeding , Molecular Sequence Data , Organ Specificity/genetics , Phenotype , RNA, Messenger/genetics , Rats , Rats, Inbred BN , Rats, Mutant Strains , Retinal Diseases/etiology , Sequence Analysis, DNA , c-Mer Tyrosine KinaseABSTRACT
We have previously reported a genetic study of a neonatal lactic acidosis linked to a pyruvate dehydrogenase complex deficiency due to the absence of the protein X subunit. This rare autosomal recessive disorder is associated with specific deletions in this polypeptide which is encoded by the HsPDX1 gene, located on chromosome 11p1.3. The pathology of the patient was considered to arise from a large homozygous deletion (78del85) found at the 5' end of the HsPDX1 coding sequence. Her heterozygous mother underwent prenatal diagnosis during a subsequent pregnancy. Chorionic villus samples were used for three independent studies: (1) normal levels of the protein X component of the PDH complex were detected by immunoblotting; (2) RT-PCR analysis showed no deletion at the 5' end of the cDNA but the presence of a distinct heterozygous deletion (965del59) at its 3' end inherited from the father; (3) haplotype analysis revealed the presence of the father's mutated allele and the mother's normal allele. It was concluded that the fetus was heterozygous for this separate 3' deletion, so, it was likely to be not affected. This study permitted us to characterize more precisely the genetic abnormalities of the HsPDX1 cDNA occurring in each family's member.
Subject(s)
Chromosome Aberrations/diagnosis , Chromosomes, Human, Pair 11 , Leigh Disease/diagnosis , Pyruvate Dehydrogenase Complex Deficiency Disease , Blotting, Western , Chorionic Villi Sampling , Chromosome Disorders , DNA Primers , DNA, Complementary/analysis , Diagnosis, Differential , Female , Gene Deletion , Haplotypes , Humans , Infant, Newborn , Mutation , Pedigree , Pregnancy , Pyruvate Dehydrogenase Complex/genetics , RNA/genetics , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We report the cystathionine-beta synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.
Subject(s)
Cystathionine beta-Synthase/genetics , Embryonic and Fetal Development/genetics , Gene Expression Regulation, Developmental , Adult , Cystathionine beta-Synthase/biosynthesis , DNA Probes , Female , Gene Expression Regulation, Enzymologic , Humans , In Situ Hybridization , PregnancyABSTRACT
Polypeptide growth factors play an important role in the regulation of human embryonic development. Insulin-like 4 gene (INSL4) is a member of the insulin family, which includes insulin, IGF-I, IGF-II, relaxin, and INSL3. Using RT-PCR, we previously found abundant INSL4 mRNA in the human placenta. In this study, we examined the chronology and spatial expression of this gene in sections of human placenta and conceptus by means of in situ hybridization. Expression of the IGF-II gene was studied as a positive control. INSL4 distribution was tissue- and cell-specific. Indeed, INSL4 mRNA was most abundant in syncytiotrophoblast cells. In fetal tissues, INSL4 mRNA was identified in the perichondrium of all four limbs, vertebrae, and ribs. Moreover, INSL4 mRNA was abundant in interbone ligaments. These findings indicate that the INSL4 gene may play an important role in trophoblast development and regulation of bone formation. IGF-II mRNA, in agreement with the literature, are mainly located in the mesodermal core in the villous trophoblast and in most embryonic tissues.
Subject(s)
Growth Substances , Intercellular Signaling Peptides and Proteins , Placenta/metabolism , Pregnancy Proteins/genetics , Trophoblasts/metabolism , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Oligonucleotide ProbesABSTRACT
Congenital microcoria is an autosomal dominant disorder characterized by a pupil with a diameter <2 mm. It is thought to be due to a maldevelopment of the dilator pupillae muscle of the iris, and it is associated with juvenile-onset glaucoma. A total genome search for the location of the congenital microcoria gene was launched in a single large family. We found linkage between the disease and markers located on 13q31-q32 (Zmax = 9.79; theta = 0). Haplotype analysis narrowed the linked region to an interval <8 cM between markers D13S1239 proximally and D13S1280 distally.
Subject(s)
Chromosomes, Human, Pair 13 , Iris/abnormalities , Chromosome Mapping , Female , Genetic Linkage , Haplotypes , Humans , Male , Pedigree , Recombination, GeneticABSTRACT
The RH blood group locus is composed of two sequence-related genes, RHD and RHCE, encoding the D, Cc, and Ee antigens in common Rh-positive phenotypes. In this report, we have analyzed the molecular basis of Rh antigens expression in weak D (Du) and RN donors, in whom there is a severe reduction of the D and C/e antigens, respectively. Genomic and transcript analysis of three unrelated low-grade weak D (Du) variants indicated that the very low expression of the D antigen is not the result of rearrangement or mutation in the coding sequence of the RHO gone. Accordingly, weak D (Du) erythrocytes should carry a normal RhD polypeptide, which is in agreement with the observation that these variants never produce anti-D antibodies. Comparative polymerase chain reaction analysis showed a lower steady-state level of RhD transcripts in weak D (Du) reticulocytes, as compared with normal RhD-positive controls, thus providing direct evidence that the difference between the D antigen of D-positive and weak D (Du) red blood cells is quantitative only. Conversely, analysis of the molecular genetic basis of the RN phenotype Indicated that the severely decreased expression of the RhC and Rhe antigens in three variants is associated with a qualitative alteration identified as a segmental DNA exchange between the RHCE and RHD genes. These genomic rearrangements, which resulted in hybrid RhCe-D-Ce proteins expressing the low frequency Rh32 but not the high incidence Rh46 antigens, involved either axon 4 alone or both exons 3 and 4. These findings show that an identical phenotypical alteration of Rh antigens (reduced expression) may result either from a quantitative or a qualitative alteration of the RH genes expression.
Subject(s)
Gene Expression Regulation , Rh-Hr Blood-Group System/biosynthesis , Rh-Hr Blood-Group System/genetics , Alleles , Base Sequence , Exons/genetics , Genetic Variation , Humans , Models, Molecular , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Recombination, Genetic , Rh-Hr Blood-Group System/chemistry , Transcription, GeneticABSTRACT
The partial D phenotypes correspond to D-positive individuals that may develop anti-D antibodies following immunization by transfusion or pregnancy, since they lack some of the D epitopes that compose the D antigen. When these red cells are tested with a panel of human monoclonal anti-D, different patterns of reactivity are observed and at least nine distinct epitopes termed epD1 to epD9 can be identified. Molecular analysis of partial D variants have shown that the loss of some D epitopes is associated either with intergenic recombination events between the D and CE genes generating hybrid gene structures D-CE-D or CE-D-CE, or with point mutations of the D gene. Based on these findings, a tentative model that correlates critical amino acid positions and D epitope expression on the D protein was proposed. Although recent studies suggest that the D antigen may be composed of as many as 30 epitopes, the relatively simple model presented here may be useful to serologists as a preliminary approach to understanding the basis of D antigenic variation in terms of structure-activity relationship.
Subject(s)
Models, Molecular , Rh-Hr Blood-Group System/genetics , Antibodies, Monoclonal , Epitopes , Humans , Peptides , Phenotype , Rh-Hr Blood-Group System/immunologyABSTRACT
The nucleotide sequence of the RhD transcripts from the reticulocytes of three unrelated variants with the DVII category blood group phenotype has been determined. Our results indicate that the expression of the low frequency antigen Rh40 and the lack of epD8 at the surface of these variant RhD positive red cells are associated with a single point mutation, T329C, in exon 2 of the RHD gene. This nucleotide polymorphism results in a leucine to proline substitution at amino acid position 110 of the RhD polypeptide.
Subject(s)
Genetic Variation , Leucine , Point Mutation , Proline , Reticulocytes/metabolism , Rh-Hr Blood-Group System/genetics , Amino Acid Sequence , Base Sequence , DNA Primers , DNA, Complementary/blood , DNA, Complementary/isolation & purification , Gene Expression , Humans , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction/methods , Protein Structure, Secondary , Rh-Hr Blood-Group System/biosynthesis , Rh-Hr Blood-Group System/chemistryABSTRACT
The RH blood group locus from RhD-positive donors is composed of two closely related genes, RHCE and RHD, encoding the Cc/Ee and D antigens, respectively. The major Rh antigen, D, is serologically defined as a mosaic of at least nine determinants (epD1 to epD9), and the lack of expression of some of these D epitopes at the surface of variant red blood cells defines the D category phenotypes. In this report, we have analyzed the Rh transcripts from reticulocytes of different D category phenotypes (DIVa, DIVb, DVa, and DFR). Although Southern blot analysis did not sow obvious deletions within the RHD gene, sequence analysis of the RhD transcripts indicated that, in all cases studied, the lack of D epitopes is associated with substitutions, in the deduced polypeptides, of amino acids specific of the RhD protein by those encoded at the equivalent position by the RHCE gene. These results strongly suggested that the D category phenotypes resulted from segmental DNA replacement between RHD-specific fragments and their equivalents in the RHCE gene. The regions involved in the DIVa, DIVb, DVa, and DFR phenotypes were shown to encompass all or part of the exons 3 and 7, exons 7 to 9, exon 5, and exon 4, respectively. All protein variants encoded by these rearranged RH genes represent new CE-D-CE hybrid molecules that retain only some of the nine D epitopes. Because segmental DNA replacements have been previously identified in other Rh variant genomes, we postulate that such genomic rearrangements between different regions of the RHCE and RHD genes should be one of the most frequent events involved in the extreme polymorphism of the RH blood group system.
Subject(s)
Rh-Hr Blood-Group System/genetics , Base Sequence , DNA Primers/chemistry , Gene Expression , Gene Rearrangement , Genes , Humans , Membrane Proteins/chemistry , Membrane Proteins/immunology , Molecular Sequence Data , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , RNA, Messenger/genetics , Rh-Hr Blood-Group System/immunologyABSTRACT
The Rh blood group antigens D, Cc and Ee are encoded by two highly related genes, RHD and RHCE. Almost all red cells which carry D and all cells which carry C also express the G (Rh12) antigen. In this report we have determined the molecular basis of the DIIIb category phenotype which represents a very rare condition characterized by the presence of most of the D epitopes and the total absence of the antigen G. mRNA sequencing and Southern blot analysis of two unrelated samples indicated that the DIIIb category phenotype is associated with a segmental DNA exchange between exon 2 of the RHD and RHCE genes resulting in three D-->c amino acid substitutions (Ile60Leu, Ser68Asn and Ser103Pro).
Subject(s)
Gene Rearrangement , Rh-Hr Blood-Group System/genetics , Base Sequence , Blotting, Southern , DNA Probes , Erythrocytes/immunology , Exons , Humans , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
The Rh (Rhesus) blood group antigens, D, Cc, and Ee, are carried by three unglycosylated membrane proteins of the human erythrocytes encoded by two highly related genes, D and CcEe. The major antigen, D, is a mosaic composed of at least nine determinants (epD1 through epD9). The lack of expression of some of these D epitopes at the surface of variant red blood cells defines the so-called D category phenotypes. In this report, we have determined the molecular basis of the DVI category phenotype characterized by the lack of epitopes D1, D2, D5, D6/7, and D8. Southern blot analysis and mRNA sequencing showed that the DVI phenotype is associated with two types of rearrangement of the D gene. Of 10 DVI genomes investigated, 8 exhibited a segmental DNA replacement (gene conversion) between the D fragment encompassing exons 4, 5, and 6 and the equivalent region of the CcEe gene. In the two other variants, these three exons are deleted. In both cases, the genomic rearrangement did not alter the reading frame of the variant RhD transcripts that are translated in 417 and 266 amino acid polypeptides, respectively. A heterogeneity of category DVI samples based on variable reactivity of the red blood cells with anti-D antibodies was previously found to be associated with the CDVIe or cDVIE haplotypes. Interestingly, our present results indicated that this serologic subdivision of the DVI category is correlated to two types of genomic rearrangements of the D gene.
Subject(s)
Gene Rearrangement , Rh-Hr Blood-Group System/genetics , Amino Acid Sequence , Base Sequence , Blotting, Southern , DNA/blood , DNA/genetics , DNA Primers , Erythrocyte Membrane/metabolism , Exons , Humans , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Restriction MappingABSTRACT
The human RH (rhesus) locus is composed of two genes, RHD and RHCE, encoding the D, Cc, and Ee blood group antigens. The RHCE gene was isolated from a human genomic library and characterized. It is organized into 10 exons distributed over 75 kb. Exons 4-8 are alternatively spliced in the different RNA isoforms previously identified. Primer extension analysis indicated that the transcription initiation site is located 83 bp upstream of the initiation codon. The 5' flanking region of the RHCE gene, from nucleotide -600 to +42, exhibited a significant transcriptional activity after transfection in the erythroleukemic cell line K562, but not in the nonhematopoietic cell line HeLa. This result was in agreement with Northern blot analysis, suggesting that the expression of the RH locus is restricted to the erythroid/megakaryocytic lineage. Accordingly, putative binding sites for SP1, GATA-1, and Ets proteins, nuclear factors known to be involved in the erythroid and megakaryocytic gene expression, were identified in this Rh promoter.