Characterisation of the murine gene encoding the intracellular hyaluronan receptor IHABP (RHAMM).

We have recently shown that the published cDNA sequence encoding the murine cell surface receptor for hyaluronan-mediated motility (RHAMM) in fact represents a partial sequence of the cDNA encoding a new intracellular hyaluronic acid binding protein (IHABP). Here we publish the genomic organisation, including 700bp sequences of the promoter region, of the IHABP gene. The IHABP gene consists of 18 exons and spans more than 25kb. Part of the IHABP gene is identical with the published data on RHAMM. The IHABP gene apparently possesses one promoter region with one major transcriptional start point. IHABP is ubiquitously expressed at the mRNA and the protein level in all murine tissues, suggesting that the function of this intracellular hyaluronan binding protein is not restricted to migrating cells.

A case of non-beta-globin gene linked beta thalassaemia in a Dutch family with two additional alpha-gene defects: the common -alpha3.7 deletion and the rare IVS1-116 (A-->G) acceptor splice site mutation.

We describe a family with beta thalassaemia, apparently not linked to the beta-globin gene cluster, in combination with alpha thalassaemia. The propositus, an adult Dutch Caucasian male, and his son presented with microcytic hypochromic parameters. Their lysates displayed the normal adult pattern on electrophoresis. The HbA2 concentration, which is usually increased in beta thalassaemia, was normal. The in vitro biosynthetic rate of the globin chains was strongly unbalanced even in the presence of a coexisting alpha-thalassaemia defect. Routine analysis of the beta genes, including the promoter region, was performed repeatedly by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGCE) and direct sequencing. No molecular abnormalities were detected. Large beta deletions were excluded by haplotype determination, using seven polymorphic markers distributed over an area of 50 kb, from 1 kb 5' of the epsilon gene to 4 kb 3' of the beta gene. The haplotype analysis of the beta-gene cluster revealed that the unaffected daughter had received the same beta haplotype as her beta-thalassaemic brother from their beta-thalassaemic father. These data suggest that the beta-gene cluster shared by father and son was not directly associated with a reduced beta-globin chain expression. In order to exclude the remote possibility of a beta-locus-control region (LCR) rearrangement in the paternal haplotype of the daughter, the sequence of the HS2 element was examined in the nuclear family. We compared the haematological and clinical data of this family with the data reported in the limited number of similar cases. We discuss the possibility that the mutation of a trans-acting erythroid factor(s), not linked to the beta-genes cluster, may impair the beta-gene expression of both alleles.

Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein.

The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.

Phenotype variability of the dominant beta-thalassemia induced in four Dutch families by the rare cd121 (G-->T) mutation.

Eight patients who were carriers of beta-thalassemia induced by the cd121 (G-->T) mutation are described in four nonrelated Dutch families. This mutant, which is considered rare and inherited in a dominant manner, is expressed in a different way among each of the four families and even among carriers of the same family. The symptoms vary from an hemolytic anemia of intermediate gravity with hepatosplenomegaly, inclusion bodies and erythroblastosis, to a mild anemia with minor hematological abnormalities. We report the analytical procedures used for the detection of the mutant, the hematological and clinical data of the four families and discuss the variable physiopathology of this molecular defect. We also compare the variation in fetal hemoglobin expression in relation to the haplotypes of the beta-gene cluster and to the different hematological conditions. The presence of this rare mutant in four nonrelated Dutch families could derive from a single mutation or from multiple events. The existence of the four mutations in three different haplotypes suggests the occurrence of at least two independent events. The presence of five abnormal hemoglobins and the beta-thalassemia defect on different haplotypes at cd121 also suggests a relatively increased rate of mutations at this particular site.

The molecular spectrum of beta-thalassemia and abnormal hemoglobins in the allochthonous and autochthonous dutch population.

The prevalence at birth of hemoglobin defects in the autochthonous North-European population is low. However, the long immigration and colonial history of the Netherlands has resulted in a group of about 1-2 million 'autochthonous' inhabitants, with Asian, South-European or African ancestors, in whom a moderate birth prevalence of globin gene mutations can be expected. Furthermore, at least 10% of the Dutch population consists of recent immigrants from different countries with high birth prevalence of hemoglobinopathies. Because of the endogamous partner choice, which is prevalent in this population, the risk for homozygous progeny remains elevated. At least 100,000 carriers of hemoglobinopathies of recent allochthonous origin are present in the Netherlands, and the number of homozygous children is rising. Prevention by prenatal diagnosis requires a suitable protocol and knowledge about the molecular defects present in the country. Therefore we have analyzed a large number of patients and carriers, both at the hematological and at the DNA level. Our survey revealed 47 different beta-thalassemia determinants, characterized on 223 independent chromosomes from individuals of different ethnic origins. As expected, the most prevalent mutations were largely represented. The cd39 (C-->T) mutation was found in 70% of the immigrants from Morocco, Sardinia and other Central-West-Mediterranean regions while the IVS-I-110 (G-->A) was prevalent in the East-Mediterranean populations. The IVS-I-5 (G-->C) mutation was found in 45% of the patients of Indonesian origin. We also registered 308 independent chromosomes with common structural defects (HbS, HbC, HbE, Hb Lepore, Hb Constant Spring and HbD Punjab) and 33 chromosomes with 19 different, less frequent, rare or very rare mutants. Seven structural mutants were described for the first time and published separately. Furthermore, 139 independent chromosomes with deletional and nondeletional alpha-thalassemia defects were characterized.

Atypical HbH disease in a Surinamese patient resulting from a combination of the -SEA and -alpha 3.7 deletions with HbC heterozygosity.

The first case of haemoglobin H (HbH) disease in combination with haemoglobin C (HbC) is reported in a man of Surinamese origin. Only haemoglobin A (HbA) and HbC were detected by electrophoresis. The amount of HbC was much less than expected in HbC heterozygotes. The synthesis ratio (beta A+ beta C/alpha) indicated an alpha-thalassaemia defect with two non-functional alpha genes, which did not correlate with the degree of haemolysis and anaemia displayed by the patient. The DNA analysis of the alpha-genes clusters revealed a defect combination -SEA/-alpha 3.7. The haematological data and the physiopathology of this atypical case are compared with the typical HbH disease found in a first cousin of the propositus. Data on the globin chains expression and on the formation of beta A and beta C homotetramers in HbH/HbC disease are presented.

Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE, PTT, and southern analysis.

Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. We screened the entire coding region of the APC gene for mutations in an unselected series of 105 Dutch FAP kindreds. For the analysis of exons 1-14, we employed the GC-clamped denaturing gradient gel electrophoresis (DGGE), while the large exon 15 was examined using the protein truncation test. Using this approach, we identified 65 pathogenic mutations in the above 105 apparently unrelated FAP families. The mutations were predominantly either frameshifts (39/65) or single base substitutions (18/65), resulting in premature stop codons. Mutations that would predict abnormal RNA splicing were identified in seven cases. In one of the families, a nonconservative amino acid change was found to segregate with the disease. In spite of the large number of APC mutations reported to date, we identified 27 novel germline mutations in our patients, which reiterates the great heterogeneity of the mutation spectrum in FAP. In addition to the point mutations identified in our patients, structural rearrangements of APC were found in two pedigrees, by Southern blot analysis. The present study indicates that the combined use of DGGE, protein truncation test, and Southern blot analysis offers an efficient strategy for the presymptomatic diagnosis of FAP by direct mutation detection. We found that the combined use of the currently available molecular approaches still fails to identify the underlying genetic defect in a significant subset of the FAP families. The possible causes for this limitation are discussed.

Hb Malmö [beta-97(FG-4)His-->Gln] leading to polycythemia in a Dutch family.

We have examined six individuals from a two-generation Dutch family for a suspected hemoglobin (Hb) abnormality. The propositus presented with polycythemia and complained of persistent weakness, headache, and epistaxis. All family members initially showed a normal Hb-electrophoretic pattern, but on isoelectric focusing, three of them displayed a fast-moving band associated with high packed red cell volumes (PCV) and increased red blood cell count. The Hb mutant was analyzed at the DNA level by specific gene fragment amplification (PCR), followed by direct DNA sequencing, and the mutation was confirmed by restriction enzyme analysis. We found a C-->G transversion (CAC-->CAG) at codon 97 of the beta-chain, which corresponded to the His-->Gln amino acid substitution previously described as Hb Malmö. We report here the clinical history of the patient, the effects of phlebotomy treatment, and the effect of subnormal iron conditions on the erythropoietic recovery after phlebotomy. The mechanism responsible for the induction of the higher oxygen affinity is discussed, as are some aspects concerning the occurrence, pathology treatment, and the genetic risk of Hb variants with high O2 affinity.

Accelerated immune response in transgenic mice expressing rat CD44v4-v7 on T cells.

Splice variants of the glycoprotein CD44 (CD44v) that confer metastatic behavior to noninvasively growing rat tumor cells are transiently expressed on lymphocytes during activation. A mAb directed against an epitope encoded by CD44 exon v6 blocks both metastasis formations and lymphocyte activation, implicating CD44v in normal immune function. To explore the nature of this function of CD44v, transgenic mice were generated that constitutively express rat CD44v4-v7 on thymocytes and peripheral T cells. The number of lymphocytes as well as the distribution of lymphocyte subpopulations were similar in nontransgenic and rat CD44v4-v7 transgenic mice. T cells of the transgenic mice, however, responded faster to activating stimuli, particularly during primary stimulations with T cell mitogens and T-dependent Ags in vivo and in vitro. This accelerated response depended on the expression of the transgene product, since a rat CD44v6-specific Ab reverted the response profiles of the transgenic mice to those of nontransgenic mice. Since the transgene gained in vivo and in vitro functional activity only upon antigenic stimulation, it is likely that CD44 variant isoforms are involved in the process of signal transduction during lymphocyte activation.

Analysis of molecular functions of the tumor metastasis promoting surface molecule CD44v4-v7 using transgenic mice.

In certain circumstances, metastatic tumor cells may mimic the molecular properties and behaviour of lymphocytes. Support for this hypothesis has come from the observation that activated lymphocytes and some tumor cells need a CD44 variant isoform (CD44v) to survive and/or expand in the lymphatic system. CD44 variant (CD44v) isoforms are created by differential splicing from a pool of at least ten variant exons (v1-v10), the encoded sequences of which are absent in the CD44 standard isoform (CD44s). To dissect the molecular interactions of CD44v, transgenic animals have been generated that constitutively express a variant of CD44 containing sequences encoded by exons v4 to v7 on the surface of T cells. Lymphocytes derived from these transgenic animals show accelerated entry into S phase upon antigenic stimulation, and a subpopulation of the cells constitutively express early lymphocyte activation markers. Our data support the hypothesis that the presence of CD44v4-v7 on the surface of T cells mediates intercellular or intracellular processes which result in the promotion of T cells towards a preactivated state.