Rh Gene Research Articles

Evolutionary history of the Rh blood group-related genes in vertebrates.

Rh and its homologous Rh50 gene products are considered to form heterotetramers on erythrocyte membranes. Rh protein has Rh blood group antigen sites, while Rh50 protein does not, and is more conserved than Rh protein. We previously determined both Rh and Rh50 gene cDNA coding regions from mouse and rat, and carried out phylogenetic analyses. In this study, we determined Rh50 gene cDNA coding regions from African clawed frog and Japanese medaka fish, and examined the long-term evolution of the Rh blood group and related genes. We constructed the phylogenetic tree from amino acid sequences. Rh50 genes of African clawed frog and Japanese medaka fish formed a cluster with mammalian Rh50 genes. The gene duplication time between Rh and Rh50 genes was estimated to be about 510 million years ago based on this tree. This period roughly corresponds to the Cambrian, before the divergence between jawless fish and jawed vertebrates. We also BLAST-searched an amino acid sequence database, and the Rh blood group and related genes were found to have homology with ammonium transporter genes of many organisms. Ammonium transporter genes can be classified into two major groups (amt alpha and amt beta). Both groups contain genes from three domains (bacteria, archaea, and eukaryota). The Rh blood group and related genes are separated from both amt alpha and beta groups.

The Analysis of Nucleotide Substitutions, Gaps, and Recombination Events between RHD and RHCE Genes through Complete Sequencing

We determined the entire nucleotide sequences of all introns within the RHD and RHCE genes by amplifying genomic DNA using long PCR methods. The RHD and RHCE genes were 57,295 and 57,831 bp in length, respectively. Aligning both genes revealed 138 gaps (insertions and deletions) below 100 bp, 1116 substitutions in all introns and all exons (coding region), and 5 gaps of over 100 bp. Homologies (%) between the RH genes were 93.8% over all introns and coding exons and 91.7% over all exons and introns. Various short tandem repeats (STRs) and many interspersed nuclear elements were identified in both genes. The proportions of Alu sequences in the RHD and RHCE genes were 25.9 and 25.7%, respectively and these Alu sequences were concentrated in several regions. We confirmed multiple recombinations in introns 1 and 2. Such multiple recombination, which probably arose due to the concentrations of Alu sequences and the high level of the homology (%), is one of most important factors in the formation and evolution of RH gene. The variability of the Rh system may be generated because of these features of RH genes. Apparent mutational hotspots and regions with low of K values (the numbers of substitutions per nucleotide site) caused by recombinations as well as true mutational hotspots may be found in human genome. Accordingly, in searching for and identifying single nucleotide polymorphisms (SNPs) especially in noncoding regions, apparent mutational hotspots and areas of low K values by recombination should be noted since the unequal distribution of SNPs will reduce the power of SNPs as genetic maker. Combining the complete sequences' data of both RH genes with serological findings will provide beneficial information with which to elucidate the mechanism of recombination, mutation, polymorphism, and evolution of other genes containing the RH gene as well as to analyze Rh variants and develop new methods of Rh genotyping.

The clinical utility of fetal cell sorting to determine prenatally fetal E/e or e/e Rh genotype from peripheral maternal blood

Objective: This study was undertaken to determine the fetal E/e or e/e Rh genotype prenatally from peripheral maternal blood by examining sorted fetal cells from alloimmunized and nonalloimmunized pregnancies. Study Design: Eighteen maternal peripheral venous blood samples were obtained before amniocentesis from 15 pregnant women who were homozygous for the e allele. Five were not alloimmunized and 10 were alloimmunized. The mononuclear cell layer was isolated from the maternal blood and enriched for fetal nucleated red blood cells by flow cytometry with monoclonal antibodies to CD36 or CD71 and to glycophorin A. Eight samples were treated with CD45 monoclonal antibody–coated magnetic beads before they were sorted to deplete the maternal sample of leukocytes (CD45+ cells). We defined the positive fetal cell fractions as the monoclonal antibody positive–sorted cells derived from the maternal samples. These included sorted cells that were CD36+/glycophorin A+, CD71+/glycophorin A+ and CD45– cells that were sorted to become CD45–/CD36+/glycophorin A+ or CD45–/CD71+/glycophorin A+. The negative fractions were the cells that were negative for either CD36/glycophorin A or CD71/glycophorin A or were the CD45+ cells. Deoxyribonucleic acid was isolated from all fractions and amplified by polymerase chain reaction with allele-specific primers for the E or e Rh genes. Gel electrophoresis was performed to detect fetal E/e or e/e Rh genotype. The fetal E/e or e/e Rh genotype was confirmed by serologic and deoxyribonucleic acid testing. The accuracy of E/e or e/e Rh genotype determination from the positive cell fractions was compared with that of E/e or e/e Rh genotype determination from the negative fractions. Results: Fetal E/e or e/e Rh genotype was determined correctly in 17 of 18 of the fetal cell enriched positive fractions (94%). Fetal E/e or e/e Rh genotype was determined correctly in 11 of 14 of the maternal samples in the negative unselected cell fractions (79%). Fetal E/e or e/e Rh genotype was determined correctly in 15 of 16 sample fractions that underwent magnetic bead separation with CD45 and were subsequently sorted into positive and negative fractions (94%). Fetal E/e or e/e Rh genotype was determined correctly in 13 of 13 of the samples obtained from the alloimmunized pregnancies (100%). Conclusions: The use of monoclonal antibodies for cell sorting or for magnetic separation predicted fetal E/e or e/e Rh genotype from peripheral maternal blood correctly in as many as 100% of alloimmunized pregnancies. Thus noninvasive fetal E/e or e/e Rh genotyping can be performed by polymerase chain reaction amplification of the rare fetal cells in maternal blood. The correct prediction of fetal E/e or e/e Rh genotype from the cell population not selected by the monoclonal antibodies suggests that there are fetal cell types other than fetal nucleated erythrocytes that can also be used as a source of fetal deoxyribonucleic acid for noninvasive genetic diagnosis. Improved technology may provide methods less laborious than cell sorting to accurately determine fetal Rh type from different fetal cell types that circulate in maternal blood. (Am J Obstet Gynecol 2000;183:462-8.)

Rhd and C/cE/e genotyping in Slovenian population

The Rhesus (Rh) blood group system is, after ABO, clinically most important. Alloantibodies directed against Rh antigens are the major cause of a haemolytic disease of newborn (HDN) and of transfusion reactions. In search for novel methods for Rh genotyping we started to compare Rh genotypes identified from different tissues and Rh phenotypes. Genotypes determined from blood samples with PCR based RhD, C/c and E/e genotyping methods were compared with serologically identified phenotypes (N=32). With two exceptions the results of phenotyping and genotyping were in concordance. Two Rh serotypes from a Slovenian family that were unexpected according to the Mendelian laws were characterised genotypically. The two family members were suspected to have a chromosomal deletion on RH gene locus.

RHD gene deletion occurred in the Rhesus box

The Rh blood group antigens derive from 2 genes,RHD and RHCE, that are located at chromosomal position 1p34.1-1p36 (chromosome 1, short arm, region 3, band 4, subband 1, through band 6). In whites, a cde haplotype with a deletion of the whole RHD gene occurs with a frequency of approximately 40%. The relative position of the 2 RH genes and the location of the RHD deletion was previously unknown. A model has been developed for the RH locus using RHD- and RHCE-related nucleotide sequences deposited in nucleotide sequence databases along with polymerase chain reaction (PCR) and nucleotide sequencing. The open reading frames of bothRH genes had opposite orientations. The 3′ ends of the genes faced each other and were separated by about 30 000 base pair (bp) that contained the SMP1 gene. The RHD gene was flanked by 2 DNA segments, dubbed Rhesus boxes, with a length of approximately 9000 bp, 98.6% homology, and identical orientation. The Rhesus box contained the RHD deletion occurring within a stretch of 1463 bp of identity. PCR with sequence-specific priming (PCR-SSP) and PCR with restriction fragment length polymorphism (PCR-RFLP) were used for specific detection of the RHDdeletion. The molecular structure of the RH gene locus explains the mechanisms for generating RHD/RHCE hybrid alleles and the RHD deletion. Specific detection of theRHD− genotype is now possible.

Evolution of RH genes in hominoids: characterization of a gorilla RHCE-like gene.

The human RH locus is responsible for the expression of the Rh blood group antigens. It consists of two closely linked genes, RHD and RHCE, that exhibit 92% similarity between coding regions. These observations suggest that they are derived from a relatively recent duplication event. Previously a study of nonhuman primate RH-like genes demonstrated that ancestral RH gene duplication occurred in the common ancestor of man, chimpanzees and gorillas. By amplification of intron 3 and intron 4 of gorilla RH-like genes, we have now shown that, like man, gorillas possess two types of RH intron 3 (RHCE intron 3 being 289 bp longer than the RHD intron 3) and two types of intron 4 (RHCE intron 4 being 654 bp longer than the RHD intron 4). Here we report the characterization of a cDNA encoded by a gorilla RH-like gene which possesses introns 3 and 4 of the RHCE type. A comparison of this gorilla RHCE-like coding sequence with previously characterized human and ape cDNA sequences suggests that RH genes experienced complex recombination events after duplication in the common ancestor of humans, chimpanzees and gorillas.

Molecular biology and genetics of the Rh blood group system

The Rh (Rhesus) blood group system is the most complex of the known human blood group polymorphisms. The expression of its antigens is controlled by a two-component genetic system consisting of RH and RHAG loci, which encode Rh30 polypeptides and Rh50 glycoprotein, respectively. Over the past decade, there has been a rapid advance in knowledge of the biochemistry, molecular biology, and genetics of the Rh genes and proteins. The primary structures of D and CcEe antigens have become well understood and the molecular genetic basis of a vast array of phenotype polymorphisms has been delineated. The identification of various molecular defects associated with Rh deficiency syndrome clarifies the nature of the amorph, suppressor, and modifier genes. The observed mutation spectrum defines a basic set of components essential for Rh complex assembly in the erythrocyte membrane. The resulting molecular information, combined with new experimental tools, is helping to dissect the fine structure of Rh antigens in terms of epitope mapping. The discovery of novel Rh homologs in primitive organisms and in nonerythroid tissues opens new avenues of research beyond the scope of erythrocytes and Rh antigens. This review provides an update on the Rh family in antigen expression, phenotype diversity, and disease association.

Gene organization and rearrangements at the human Rhesus blood group locus revealed by fiber-FISH analysis.

The human Rhesus (Rh) blood group locus is composed of two highly homologous genes, the RHD and RHCE genes on chromosome 1, encoding the D, C/c, and E/e antigens in common Rh-positive phenotypes. In general, the RHD gene is either absent or grossly deleted in Rh-negative individuals. In this study, gene organization at the RH locus of Japanese donors with different serological phenotypes was directly analyzed by two-color fluorescence in situ hybridization on DNA fibers released from their lymphocytes (fiber-FISH) and by using DNA probes of introns 3 and 7 of the RHCE and RHD genes. Six Rh-positive samples (two with the D+C-c+E+e-, two with the D+C+c-E-e+, and two with the D+C+c+E+e+ phenotype) showed the presence of two RH genes within a region of less than 200 kb on chromosome 1p36.1. Of great interest was the finding that the genes were arranged in the antidromic order of the telomere -RHCE (5'--> 3') -RHD (3'-->5') - centromere. On the other hand, two typical Rh-negative samples (D-C-c+E+e+) showed the presence of only one RHCE gene, as expected. Moreover, further analysis combined with a locus-specific assay of three Rh-negative samples (D-C+c+E+e+, D-C+c+E-e+, and D-C+c-E-e+) showed the possible presence of the RHD gene(s) and complex rearrangements, including partial deletion, duplication, and recombination, in this region; these could be responsible for the Rh-negative phenotype.

Rh gene evolution in primates: study of intron sequences.

By amplification and sequencing of RH gene intron 4 of various primates we demonstrate that an Alu-Sx-like element has been inserted in the RH gene of the common ancestor of humans, apes, Old World monkeys, and New World monkeys. The study of mouse and lemur intron 4 sequences allowed us to precisely define the insertion point of the Alu-Sx element in intron 4 of the RH gene ancestor common to Anthropoidea. Like humans, chimpanzees and gorillas possess two types of RH intron 4, characterized by the presence (human RHCE and ape RHCE-like genes) or absence (human RHD and ape RHD-like genes) of the Alu-Sx element. This led us to conclude that in the RH common ancestor of humans, chimpanzees, and gorillas, a duplication of the common ancestor gene gave rise to two genes, one differing from the other by a 654-bp deletion encompassing an Alu-Sx element. Moreover, most of chimpanzees and some gorillas posses two types of RHD-like intron 4. The introns 4 of type 1 have a length similar to that of human RHD intron 4, whereas introns 4 of type 2 display an insertion of 12 bp. The latest insertion was not found in the human genome (72 individuals tested). The study of RH intron 3 length polymorphism confirmed that, like humans, chimpanzees and gorillas possess two types of intron 3, with the RHD-type intron 3 being 289 bases shorter than the RHCE intron 3. By amplification and sequencing of regions encompassing introns 3 and 4, we demonstrated that chimpanzee and gorilla RH-like genes displayed associations of introns 3 and 4 distinct to those found in man. Altogether, the results demonstrate that, as in humans, chimpanzee and gorilla RH genes experienced intergenic exchanges.

Rhd and C/cE/e genotyping in Slovenian population.

The Rhesus (Rh) blood group system is, after ABO, clinically most important. Alloantibodies directed against Rh antigens are the major cause of a haemolytic disease of newborn (HDN) and of transfusion reactions. In search for novel methods for Rh genotyping we started to compare Rh genotypes identified from different tissues and Rh phenotypes. Genotypes determined from blood samples with PCR based RhD, C/c and E/e genotyping methods were compared with serologically identified phenotypes (N=32). With two exceptions the results of phenotyping and genotyping were in concordance. Two Rh serotypes from a Slovenian family that were unexpected according to the Mendelian laws were characterised genotypically. The two family members were suspected to have a chromosomal deletion on RH gene locus.

RH blood group system and molecular basis of Rh-deficiency

Rhesus (Rh) antigens are defined by a complex association of membrane polypeptides that are missing or severely deficient from the red cells of rare Rhnullindividuals who suffer a clinical syndrome of varying severity characterized by abnormalities of the red cell shape, cation transport and membrane phospholipid organization. The Rhnullphenotype is an inherited condition that may arise from homozygosity either for a ‘suppressor’ gene unrelated to the RH locus (‘regulator type’) or for a silent allele at the RH locus itself (‘amorph type’).A current model suggests that the proteins of the Rh complex (Rh, RhAG, CD47, LW, GPB) are assembled by non-covalent bonds and that it is not assembled or transported to the cell surface when one subunit is missing. Rh and RhAG proteins belong to the same protein family and are quantitatively the major components that form the core of the complex, which is firmly linked to the membrane skeleton. Molecular analysis of Rhnullindividuals has revealed that abnormalities occur only at the RHAG and RH loci, without alteration of the genes encoding the accessory chains.Mutations of the RHAG gene, but not of RH, occur in all Rhnullindividuals of the regulator type (including Rhmod) investigated so far (13 cases), strongly suggesting thatRHAG mutants act as ‘suppressors’ and not as transcriptional regulators of the RH genes and that variable expression of the RHAG alleles may account for the Rhmodphenotypes (exhibiting weak expression of Rh antigens). Conversely, mutations of the RHCE gene, but not of RHAG, occur in two unrelated Rhnullindividuals of the amorph type, supporting the view that RH mutants result from a ‘silent’ allele at the RH locus. These findings strongly support the Rh complex model since when either the Rh or RhAG protein is missing, the assembly and/or transport of the Rh complex is defective. Transcriptional as well as post-transcriptional mechanisms may account for the molecular abnormalities, but experimental evidence based on expression models is required to test these hypotheses, in the hope that they may help to clarify the biological role of the Rh proteins in the red cell membrane.

Evidence for a separate genetic origin of the partial D phenotype DBT in a Japanese family.

In a Moroccan family, the partial D phenotype DBT is defined by an RHD-CE-D gene in which exons 5 to 7 of RHD were replaced by those of RHCE. In this study, the molecular basis and inheritance of DBT in a Japanese family are described. A Japanese proposita exhibiting the DBT phenotype was analyzed by serologic methods and molecular techniques. The RH transcripts of the proposita were sequenced and compared with those of normal donors. The inheritance and structure of the RH genes in the family were determined by Southern blot analysis and exon-specific polymerase chain reaction. The proposita typed weak D and C+c+E+e+Rh:32. Family data indicated a cotransmission of Rh32 with DBT and a linkage of C and e with DBT. Southern blot testing of the proposita's genomic DNA indicated a partial and a total absence of RHD on the respective homologous chromosomes. Sequencing of her cDNA showed expression of Ce, cE, and RHD-CE-D transcripts but not D. The RHD-CE-D hybrid was characterized by a conversion of five RHD exons into RHCE exons (exons 5-9). The 5' and 3' breakpoints in the fusion gene were localized to the intron 4/exon 5 region and intron 9. The new RHD-CE-D gene defines a separate genetic origin of DBT in the Japanese family. The proximal sequence encoded by RHD exon 4 and RHCE exon 5, together with the distal RHCE sequence, may involve the cotransmission of Rh32 and DBT which behave as codominant and recessive characters, respectively.

The members of the RH gene family ( RH50 and RH30) followed different evolutionary pathways : Matassi G, Chérif-Zahar B, Pesole G, et al. J Mol Evol 48:151–159, 1999

The members of the RH gene family ( RH50 and RH30) followed different evolutionary pathways : Matassi G, Chérif-Zahar B, Pesole G, et al. J Mol Evol 48:151–159, 1999

Sequences and evolution of mammalian RH gene transcripts and proteins : Apoil PA, Blancher A. Immunogenetics 49:15–25, 1999

Sequences and evolution of mammalian RH gene transcripts and proteins : Apoil PA, Blancher A. Immunogenetics 49:15–25, 1999

Sequence Analysis of the Spacer Region between the RHD and RHCE Genes

Numerous variants of the Rh blood group system, discovered by Levine and Stetson in 1939, have been detected and more than forty antigens have been identified. By performing the molecular genetic analysis of the introns as well as the exons in both RH genes, it was elucidated that Rh variants were generated by gene conversion or recombination, deletions, or mutations. For understanding the generation of many Rh variants and Rh antigens in detail, it is necessary to analyze not only the RHCE and RHD genes but also the structure and the physical distance between both these RH genes. In order to achieve the aforesaid purpose, the spacer region between the RHD and RHCE genes were amplified by the long PCR method. Therefore the full spacer region was determined to be 12159 bp in length and contained the Alu consensus sequences and the putative CpG island. It was probable that the duplication of both RH genes occurred within about 12 kb region. Analysis of the spacer region provides new information for the research on the transcription-control region, the molecular evolution of RH genes, Rh variants, and the deletion of the RHD gene in Rh blood group system.

Application of RHD and RHCE genotyping for correct blood group determination in chronically transfused patients.

In chronically transfused patients, conventional blood group typing may be impossible because of mixed-field agglutination. In 27 patients with congenital anemia and lifelong transfusion history, genotyping for D, RHD, and RHCE was performed with polymerase chain reactions. These results were compared with the blood group typing results documented in the medical record. Two of 27 cases had been typed D-negative by serologic tests and D-positive by genotyping. In 20 patients, the CDE formula had been determined serologically according to the medical record; 4 of these patients were Cc by serologic tests and C/C by genotyping. One patient typed ee by serologic tests, and genotyping revealed heterozygosity (E/e). In patients with a lifelong transfusion history, serologic blood group determination may be impossible, and pretransfusion test results are not always available or reliable. In whites, Rh-matched transfusions are possible with genotyping. The genetic background of the RH genes has to be elucidated in other ethnic groups, such as in black patients with sickle cell disease, before genotyping can be applied without restriction.

The members of the RH gene family (RH50 and RH30) followed different evolutionary pathways.

The evolution of the RH gene family is characterized by two major duplication events, the first one originating the RH50 and RH30 genes and the second one giving rise to RHCE and RHD, the two paralogous RH30 genes which encode the Rh blood group antigens in human. The new sequence data obtained here for mouse RH50 and RH30 and for macaque RH50 allowed us to compare the evolutionary rates of the two genes and to show that RH50 evolved about 2.6 times more slowly than RH30 at nonsynonymous positions. This result implies that Rh50 proteins were evolutionarily more conserved compared to Rh30 polypeptides, thus being indicative of the functional significance of the former protein in species as distantly related as sponge and human. The duplication event leading to RH50 and RH30 genes was estimated to have occurred between 250 and 346 million years ago. Moreover, we could also estimate that the duplication event producing the RHCE and RHD genes occurred some 8.5 +/- 3.4 million years ago, in the common ancestor of human, chimpanzee, and gorilla. Interestingly, this event seems to coincide with the appearance in these species of a G-to-T mutation in the RH50 gene which created a stop codon in the corresponding transcript. This led to an Rh50 C-terminal cytoplasmic domain shorter than that found in orangutan and early primates.

The mouse Rhl1 and Rhag genes: sequence, organization, expression, and chromosomal mapping.

To seek an alternative model for studies of the Rh protein complex, we isolated by homology cloning and characterized the mouse Rhced and Rhag genes, which are homologous to the human RH and RHAG genes, respectively. Rhced encodes a glycoprotein of 418 amino acids which occurs as a composite of human RhD and RhCE with 60% identity and 74% similarity. Rhag encodes a glycoprotein of 438 amino acids that shares 79% identity and 87% similarity to human Rh50. However, Rhag has an elongated C terminus and four N-glycosylation sites clustered on exoloop 1. Hydropathy plots suggest that Rhl1 and Rhag each span the lipid bilayer 12 times, with N and C termini facing the cytoplasm. Rhced and Rhag are both specified by 10 exons and bear a similar exon/intron structure, but their major transcription start sites are mapped at -17A and -27A. Northern analysis revealed coexpression of Rhced and Rhag from 11-day embryos throughout adult life in erythroid tissues. Southern blotting and linkage analysis showed that Rhced and Rhag are single-copy genes localized to chromosomes 4 and 17, respectively; they are paralogous to one another but orthologous to human RH and RHAG. The results together predate the occurrence and signify a conserved function of the erythroid-specific Rh membrane structures.

Gorilla RH-like genes and antigens.

It has been previously shown that most of the human IgG monoclonal D-specific antibodies define a polymorphism in the gorilla consisting of two phenotypes: Dgor-positive and Dgor-negative. By quantitative indirect immunofluorescence assay and quantitative immunoblotting it was evaluated that the number of Dgor antigenic sites per gorilla red cell varies from a level equivalent to that observed for human RhD-positive cells to a level eight times higher. By immunoblotting with a rabbit reagent specific for the carboxylic end of human Rh-polypeptides it was demonstrated that RBCs from all gorillas, whatever their Dgor phenotype, possess 33000 relative molecular mass Rh-like polypeptides. The expression of the Dgor antigen was shown to be associated with the presence of three polymorphic bands defined by Southern blot using a human exon 4 RHCE probe, and to a length polymorphism of gorilla intron 3 evidenced by polymerase chain reaction. By contrast, the expression of the Dgor antigen was not associated to the length polymorphism of gorilla intron 4 which is related to the presence or absence of an Alu-Sx element in intron 4, paralleling the situation observed in human. These results confirmed the presence in the gorilla genome of at least two RH-like genes, one of which being responsible for Dgor polymorphism. The phylogenesis of the human and gorilla RH genes is discussed in light of the comparison of intron 4 sequences.

MDM2 amplification and loss of heterozygosity at Rb and p53 genes: no simultaneous alterations in the oncogenesis of liposarcomas.

The present study aimed to investigate the status of alterations of the MDM2, Rb and p53 genes in a series of 45 liposarcomas. Furthermore, the possible correlation with histological and clinical parameters was studied. MDM2 amplification was examined by non-radioactive Southern blot hybridization with a human MDM2 cDNA probe. Mutations in the p53 gene were screened by polymerase chain reaction/single-strand conformation polymorphism analysis and direct sequencing. To study loss of heterozygosity (LOH) at the tumor-suppressor genes Rb and p53, we used four polymorphic intragenic Rb markers (introns 1, 17, 20, and 25) and two p53 markers (intron 1 and exon 4). MDM2 amplification was found in 19 of 45 liposarcomas (42.2%). The frequency of LOH in Rb and p53 was nearly identical (22%). In 4 of 9 tumors (44.4%) with LOH, allelic loss was a concurrent event in both genes. Of 45 liposarcomas, 6 (13.3%) showed p53 mutations. Overall, alterations of the p53/MDM2/Rb pathway occurred in 30 of 45 liposarcomas (66.6%). In contrast to myxoid and pleomorphic variants, well-differentiated liposarcomas were characterized by a high frequency of MDM2 amplification, a lack of LOH of Rb and p53, and p53 mutations. Obviously MDM2 amplification and LOH at the Rh and p53 genes do not occur simultaneously in the oncogenesis of liposarcomas, as is the case for MDM2 amplification and p53 gene mutations (with one exception). We suggest that well-differentiated, myxoid and pleomorphic liposarcomas are characterized by a different pattern of molecular alterations.