High resolution mapping of OCA2 intragenic rearrangements and identification of a founder effect associated with a deletion in Polish albino patients

Oculocutaneous albinism type 2 (OCA2) represents about 30% of OCA worldwide. Using quantitative multiplex fluorescent PCR and very high-resolution array-CGH focussed on the OCA2 gene and surrounding regions in 15q12, we identified new rearrangements. Deletion 1, encompassing exons 3-20, was present in three patients (including one in the homozygous state), and Deletion 2 (exons 1-20) was found in one patient (heterozygous state). The duplication (exons 3-20) was found in one patient in the homozygous state. Using 14 microsatellite markers we determined haplotypes associated with these rearrangements. Deletion 1 was associated with the same haplotype in three patients who were all of Polish origin, which is strongly in favour of a founder effect. Deletion 2 was associated with a distinct haplotype. The homozygous duplication was inherited from the two unrelated parents of the patients on two different haplotypes. Analysis of the sequences around the breakpoints of these rearrangements showed that all occurred within complex arrays of repetitive sequences. The combined use of very high-resolution array-CGH and of microsatellites (including new intragenic ones described here) constitutes a powerful approach for the precise characterization of OCA2 rearrangements, which have been found in more than 20% of OCA2 patients.     

Chronic heart failure selectively induces regional heterogeneity of insulin-responsive glucose transporters

Glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (GLUTs), which includes GLUT4 (the major cardiac isoform) and GLUT12 (a novel, second insulin-sensitive isoform). Potential regional patterns in glucose transport across the cardiac chambers have not been examined; thus, we hypothesized that insulin-responsive GLUT4 and -12 protein and gene expression would be chamber specific in healthy subjects and during chronic heart failure (HF). Using a canine model of tachypacing-induced, progressive, chronic HF, total GLUT protein and messenger RNA in both ventricles and atria (free wall and appendage) were investigated by immunoblotting and real-time PCR. In controls, GLUT4, but not GLUT12, protein content was significantly higher in the atria compared with the ventricles, with the highest content in the right atrium (RA; P < 0.001). GLUT4 and GLUT12 mRNA levels were similar across the cardiac chambers. During chronic HF, GLUT4 and GLUT12 protein content was highest in the left ventricle (LV; by 2.5- and 4.2-fold, respectively, P < 0.01), with a concomitant increase in GLUT4 and GLUT12 mRNA (P < 0.001). GLUT4, but not GLUT12, protein content was decreased in RA during chronic HF (P = 0.001). In conclusion, GLUT4 protein was differentially expressed across the chambers in the healthy heart, and this regional pattern was reversed during HF. Our data suggest that LV was the primary site dependent on both GLUT4 and GLUT12 during chronic HF. In addition, the paradoxical decrease in GLUT4 content in RA may induce perturbations in atrial energy production during chronic HF.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Immune oxidative injury induced in mice exposed to normobaric O2: effects of thiol compounds on the splenic cell sulfhydryl content and Con A proliferative response

In vivo exposure of mice to normobaric O2 depresses the cellular immune response by a mechanism that remains unknown. In vitro oxidative injury leads to decreased sulfhydryl groups (SH) in lymphocytes. To determine whether in vivo exposure to O2 would have similar effects, we measured the SH content in spleen cells both from mice that had been exposed to normobaric O2 (O2 SC) and from controls exposed to ambient air (Air SC). The SH content of the fresh O2 SC was slightly decreased, whereas after 48 hr of culture, the SH content and the proliferative response of these cells were found to vary with the type and concentration of thiol or disulfide compounds added to the culture medium. Under standard culture conditions, i.e., RPMI 1640 medium containing 0.41 mM half-cystine, the SH content in O2 SC decreased sharply to about 10 and 20% that of Air SC in the absence or presence of Con A (2 micrograms/ml), respectively. Under these culture conditions, the proliferative response of O2 SC was 20.5% +/- 3.2 of Air SC. In cystine-free RPMI 1640 medium supplemented with various concentrations of L-cystine, L-cystine and 2-mercaptoethanol (2-ME), L-cysteine, or reduced glutathione (GSH), the proliferative response to Con A and the SH content of the O2 SC varied in parallel and were correlated (p less than 0.01). Half-cystine (0.41 mM) plus 2-ME (5 X 10(-5) M) or L-cysteine alone (4 mM) completely protected the SH content of O2 SC and induced a proliferative response 82% +/- 6 that of the controls. In cystine-free RPMI 1640 medium supplemented with GSH (4 mM), the SH content and proliferative response of O2 SC were 79 and 67.5% of Air SC, respectively. Other concentrations of these compounds were less effective. Oxygen scavengers such as SOD, catalase, mannitol, and vitamin E did not protect against the decrease of the O2 SC. The induced oxidative cellular damage might be related in part to a membrane lipid peroxidative process. These data show that in vivo exposure of mice to normobaric O2 induced lesions in splenic cells manifested under standard culture conditions by a decrease in both SH content and Con A proliferative response. The extent of these alterations could be modulated by variations of the thiol environment. Protection of the SH content correlated with protection of the proliferative response of the O2 SC.     

Analysis of the expression of the oncogene c-myc in human breast adenocarcinoma

The c-myc oncogene was characterized and its expression analyzed in 32 mammary adenocarcinomas and in 2 benign breast tumors from 34 untreated patients. Southern blot hybridization experiments have demonstrated the amplification of the oncogene (3 to 30 fold) in 3 carcinomas. The analysis of total RNA by Northern blot revealed the presence of a 2.4 kb c-myc RNA band. In 7 out of 10 carcinomas from patients with 3 or more than 3 lymph node metastases the level of c-myc expression evaluated by dot blot analysis was 4 to 14 fold greater than that of normal human tissues. In only 5 out of 22 carcinomas from patients without lymph node metastases or less than 3 invaded nodes the level of c-myc expression was also higher (4 to 10 fold). The level of c-myc expression was not significantly enhanced in the 2 benign tumors. It is suggested that the c-myc gene activation could be associated to a higher degree of malignancy of mammary carcinomas.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.      

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.     

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.