Numerous linkage studies have indicated chromosome 18q21-22 as a locus of importance for blood pressure regulation. This locus harbors the neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) gene, which is instrumental for the regulation of the amiloride-sensitive epithelial sodium channel (ENaC). In a linkage study of 16 markers (including two single nucleotide polymorphism markers located within the NEDD4L gene) on chromosome 18 between 70-104 cM and ambulatory blood pressure (ABP), in 118 families, the strongest evidence of linkage was found for 24 h and day-time systolic ABP at the NEDD4L locus (82.25 cM) (P=0.0014). In a large population sample (n=4001), we subsequently showed that a NEDD4L gene variant (rs4149601), which by alternative splicing leads to varying expression of a functionally crucial C2 domain, was associated with diastolic blood pressure (DBP) (P=0.03) and DBP progression over time (P=0.04). A genotype combination of the rs4149601 and an intronic NEDD4L marker (rs2288774) was associated with systolic blood pressure (SBP) (P=0.01), DBP (P=0.04), and progression of both SBP (P=0.03) and DBP (P=0.05) over time. A quantitative transmission disequilibrium test in the family material of the rs4149601 supported this NEDD4L variant as being at least partially causative of the linkage result. In conclusion, our findings suggest that the chromosome 18 linkage peak at 82.25 cM is explained by genetic NEDD4L variation affecting cross-sectional and longitudinal blood pressure, possibly as a consequence of altered NEDD4L interaction with ENaC.
The aim of the present work was to investigate whether acute myeloblastic leukaemia (AML) blast cells express a soluble (s) form of interleukin 6 (IL-6) receptor (R), and if they do, what is the mechanism of production. Eight AML patient cell lines and 25 primary AML blast cell samples were investigated. The cell lines secreted high quantities of sIL-6R into their culture medium when examined by enzyme-linked immunosorbent assay (ELISA). To determine whether sIL-6R is synthesized by a mechanism of alternative splicing, RNA was analysed from all the AML blast cell samples by using reverse transcription polymerase chain reaction. In this method, primer sites flanking the transmembrane domain were utilized and the alternatively spliced IL-6R mRNA was distinguished from the non-spliced transcript form by size. All the cell lines and 64% of the primary blast cell samples expressed the alternatively spliced IL-6R mRNA. To confirm the phenomenon of alternative splicing at protein level, cytoplasmic protein fractions of the cell lines were investigated by using a sensitive adaptation of the Western blot method. All the cell lines expressed two IL-6R proteins sized 80 and 50 kDa and corresponding to the membraneous and soluble forms of IL-6R, respectively. In conclusion, the results obtained at both mRNA and protein levels strongly support alternative splicing as a mechanism of sIL-6R production in AML. Because sIL-6R modulates the effects of IL-6 on target cells, differences in sIL-6R expression levels may partially explain the previously observed diversity in IL-6-induced growth responses in AML
In order to identify patients at risk for developing post-transplant lymphoproliferative disease (PTLD), a sensitive nested RT-PCR method for detection of EBNA1 gene expression in peripheral blood cells was used. EBNA1 expression in peripheral blood samples from 60 organ recipients was analyzed and compared with 24 healthy controls in a retrospective study. Overall, EBNA1-positive samples were detected at least once in 43% of the transplant patients with post-transplant lymphoproliferative disease, in 18% of the other transplant patients and in none of the healthy controls. The odds ratio for EBNA1 expression in patients with post-transplant lymphoproliferative disease was 3.42 (95% CI=1.02-11.54) compared to other transplant recipients. Together with normal EBV Q promoter initiated EBNA1 transcripts, an alternatively spliced form was expressed in peripheral blood cells in the above-mentioned transplant patients. This transcript lacks the U leader exon in the 5'-untranslated region (UTR). We have previously identified and characterized a functional internal ribosome entry site, the EBNA IRES, in the untranslated U leader exon of EBNA1. Transfection experiments with EBNA1 coding plasmids followed by Western blot showed that the EBNA IRES promotes cap-independent translation and increases the EBNA1 protein level. The alternative EBNA1 transcript lacking this function is expressed in the majority of the investigated EBNA1-positive patient samples as well as in some EBV-positive B-cell lines. Alternative splicing in this form gives EBV potential to regulate the translation of EBNA1 by modifying the 5' UTR. These findings indicate a new mechanism for EBNA1 expression in vivo.
Expression of mRNA for interleukin-6, interleukin-6Delta3, and interleukin-6Delta5 was detected in placental tissue (second and third trimesters of pregnancy) and spleen of mice immunized with sheep erythrocytes in high dose. We hypothesize that translation of mRNA yields proteins capable of binding to individual subunits of the interleukin-6 receptor and possessing effector functions.
Otospondylomegaepiphyseal dysplasia (OSMED) is an autosomal recessive skeletal dysplasia accompanied by severe hearing loss. The phenotype overlaps that of the autosomal dominant disorders-Stickler and Marshall syndromes-but can be distinguished by disproportionately short limbs, severe hearing loss, and lack of ocular involvement. In one family with OSMED, a homozygous Gly-->Arg substitution has been described in COL11A2, which codes for the alpha2 chain of type XI collagen. We report seven further families with OSMED. All affected individuals had a remarkably similar phenotype: profound sensorineural hearing loss, skeletal dysplasia with limb shortening and large epiphyses, cleft palate, an extremely flat face, hypoplasia of the mandible, a short nose with anteverted nares, and a flat nasal bridge. We screened affected individuals for mutations in COL11A2 and found different mutations in each family. Individuals from four families, including three with consanguineous parents, were homozygous for mutations. Individuals from three other families, in whom parents were nonconsanguineous, were compound heterozygous. Of the 10 identified mutations, 9 are predicted to cause premature termination of translation, and 1 is predicted to cause an in-frame deletion. We conclude that the OSMED phenotype is highly homogenous and results from homozygosity or compound heterozygosity for COL11A2 mutations, most of which are predicted to cause complete absence of alpha2(XI) chains.
The peculiarities of alternative CD44 mRNA splicing in thyroid cancer tissue of children from radiocontaminated areas was investigated. CD44 gene expression in thyroid cancer tissues of children exposed to radiation resembled that in spontaneously emerged cancers. It was concluded that CD44 gene expression is not the primary target of radioactive irradiation. Probably, the CD44 mRNA splicing deregulation is the consequence of cancer.
Susceptibility to asthma depends on variation at an unknown number of genetic loci. To identify susceptibility genes on chromosome 7p, we adopted a hierarchical genotyping design, leading to the identification of a 133-kilobase risk-conferring segment containing two genes. One of these coded for an orphan G protein-coupled receptor named GPRA (G protein-coupled receptor for asthma susceptibility), which showed distinct distribution of protein isoforms between bronchial biopsies from healthy and asthmatic individuals. In three cohorts from Finland and Canada, single nucleotide polymorphism-tagged haplotypes associated with high serum immunoglobulin E or asthma. The murine ortholog of GPRA was up-regulated in a mouse model of ovalbumin-induced inflammation. Together, these data implicate GPRA in the pathogenesis of atopy and asthma.
The Ataxia Telangiectasia Mutation (ATM) gene is mutated in the rare recessive syndrome Ataxia Telangiectasia (AT), which is characterized by cerebellar degeneration, immunodeficiency, and cancer predisposition. In this study, 41 AT families from Denmark, Finland, Norway, and Sweden were screened for ATM mutations. The protein truncation test (PTT), fragment length and heteroduplex analyses of large (0.8-1.2 kb) cDNA fragments were used. In total, 67 of 82 (82%) of the disease-causing alleles were characterized. Thirty-seven unique mutations were detected of which 25 have not previously been reported. The mutations had five different consequences for the ATM transcript: mutations affecting splicing (43%); frameshift mutations (32%); nonsense mutations (16%); small in-frame deletions (5%); and one double substitution (3%). In 28 of the probands mutations were found in both alleles, in 11 of the probands only one mutated allele was detected, and no mutations were detected in two Finnish probands. One-third of the probands (13) were homozygous, whereas the majority of the probands (26) were compound heterozygote with at least one identified allele. Ten alleles were found more than once; one Norwegian founder mutation constituted 57% of the Norwegian alleles. Several sequence variants were identified, none of them likely to be disease-causing. Some of them even involved partial skipping of exons, leading to subsequent truncation of the ATM protein.
Characterization of the spectrum of alternative splicing of alpha 1 (XIII) collagen transcripts in HT-1080 cells and calvarial tissue resulted in identification of two previously unidentified alternatively spliced sequences, one previously unidentified exon, and nine new mRNA variants.
Amplification of a COL1-encoding region of alpha 1 (XIII) collagen transcripts of HT-1080 cell RNA suggested that exon 3 of the alpha 1 (XIII) collagen gene, which was previously deduced to be of 35 base pairs (bp) may consist of a constitutive 8-bp exon and an alternatively spliced 27-bp exon, termed here exons 3A and 3B, respectively. Furthermore, a previously unidentified alternatively spliced Gly-Xaa-Yaa-encoding exon designated as 4B was found between the sequences encoded by exons 4, redesignated here as 4A and 5. Six of the 16 potential combinations of the four consecutive alternatively spliced exons 3B, 4A, 4B, and 5 were found to exist in mRNAs, and as a result, the length of the COL1 domain may vary between 57 and 104 amino acid residues. Most of the NC2 domain is encoded by the alternatively spliced exons 12 and 13. Where previous analysis of cDNAs indicated that mRNA variants exist that contain either exon 12 or 13 sequences, amplification studies indicated here that there are also variants that lack both exons 12 and 13 but none that contain both exons simultaneously. Thus, the predicted length of this domain is either 12, 31, or 34 residues. Analyses covering both the COL1 and NC2 domains demonstrate that at least 12 mRNA species exist through the alternations of exons 3B-5, 12, and 13.