Regional chromosomal assignment of the human glucocorticoid receptor gene to 5q31

Summary The gene for the human glucocorticoid receptor, previously mapped to chromosome 5, has been further localised to 5q31 by in situ hybridisation using a biotinylated 4.3-kb cDNA probe.     

Faithful expression of the human 5q31 cytokine cluster in transgenic mice

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.     

Association between a new 3q;5q chromosomal translocation and dystrophy of human retinal pigment epithelium

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degeneration. This group of disorders essentially leads to blindness due to mutations in different genes. The genetic basis affected by sporadic and inherited autosomal dominant, autosomal recessive or X-linked mutations is complex. In humans, RP is in most cases associated with missense mutations in the rhodopsin gene (RHO). RHO plays an important role in phototransduction pathways. So far, few studies have described associations between chromosomal alterations and RP. In this study, we present a case report of a premature, 32-week-old male baby who suffered from retinopathy, facial dysmorphisms and other disorders. His chromosomes were analyzed by conventional and high-resolution chromosomal techniques. This analysis revealed structural aberrations on chromosomes 3 and 5 with an apparently balanced chromosomal translocation with karyotype 46,XY,t(3;5)(q25;q11.2). Remarkably, the 3q breakpoint on the long arm of chromosome 3 is located close to the physical RHO chromosomal gene location. In this study, we describe presumably for the first time a possible association between a 3q;5q chromosomal alteration and RP. We conclude that the new detected chromosomal translocation may lead either to loss or inactivation of the intragenic RHO gene or its respective gene regulatory region. As a consequence, the chromosomal aberration may be responsible for retinitis pigmentosa.     

Familial eosinophilia maps to the cytokine gene cluster on human chromosomal region 5q31-q33.

Familial eosinophilia (FE) is an autosomal dominant disorder characterized by peripheral hypereosinophilia of unidentifiable cause with or without other organ involvement. To localize the gene for FE, we performed a genomewide search in a large U.S. kindred, using 312 different polymorphic markers. Seventeen affected subjects, 28 unaffected bloodline relatives, and 8 spouses were genotyped. The initial linkage results from the genome scan provided evidence for linkage on chromosome 5q31-q33. Additional genotyping of genetic markers located in this specific region demonstrated significant evidence that the FE locus is situated between the chromosome 5q markers D5S642 and D5S816 (multipoint LOD score of 6.49). Notably, this region contains the cytokine gene cluster, which includes three genes-namely, those for interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF)-whose products play important roles in the development and proliferation of eosinophils. These three cytokine genes were screened for potential disease-specific mutations by resequencing of a subgroup of individuals from the present kindred. No functional sequence polymorphisms were found within the promoter, the exons, or the introns of any of these genes or within the IL-3/GM-CSF enhancer, suggesting that the primary defect in FE is not caused by a mutation in any one of these genes but, rather, is caused by another gene in the area.     

Intralysosomal cystine accumulation in mice lacking cystinosin,the protein defective in cystinosis

Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.     

Bap31 is a novel target of the human papillomavirus E5 protein

The E5 proteins of human papillomaviruses (HPVs) are small hydrophobic proteins that are expressed in the early and late stages of the viral life cycle; however, their role in HPV pathogenesis is not clearly understood. In this study, a split-ubiquitin yeast (Saccharomyces cerevisiae) two-hybrid system was used to identify B-cell-associated protein 31 (Bap31) as a binding partner of HPV E5 proteins. The association of these proteins was confirmed by coimmunoprecipitation of complexes of Bap31 with either HPV type 16 (HPV16) or HPV31 E5. In addition, Bap31 and E5 were found to colocalize in perinuclear patterns consistent with localization to the endoplasmic reticulum. Mutational analysis of E5 identified amino acids in the extreme C terminus as important for stabilizing the interaction with Bap31. Deletion of these C-terminal amino acids of E5 in the context of complete HPV31 genomes resulted in impaired proliferative capacity of HPV-positive keratinocytes following differentiation. When small interfering RNAs were used to reduce the levels of Bap31, the proliferative ability of HPV-positive keratinocytes upon differentiation was also reduced, implicating Bap31 as a regulator of this process. These studies identify a novel binding partner of the high-risk HPV E5 proteins and provide insight into how the E5 proteins may modulate the life cycle in differentiating cells.     

Evidence in favor of linkage to human chromosomal regions 18q, 5q and 13q for bicuspid aortic valve and associated cardiovascular malformations

The aim of this study was to identify regions of the genome that harbor genes influencing inheritance of bicuspid aortic valve (BAV) and/or associated cardiovascular malformation (CVM). Aortic valve disease is an important clinical problem, which often results in valve replacement, the second most common cardiac surgery in the United States. In every age group, a majority of cases of valve disease involves a BAV. BAV is the most common CVM with a reported prevalence of 1–2%. Heritability studies indicate that BAV determination is almost entirely genetic. We used a family-based genome-wide linkage analysis with microsatellite markers. Parametric and nonparametric analyses were performed with the software GENEHUNTER and SOLAR (Sequential Oligogenic Linkage Analysis Routines). Thirty-eight families (353 subjects) with BAV and/or associated CVM were assessed. Each participant underwent a standardized echocardiographic examination. The highest LOD score, 3.8, occurred on chromosome 18q between markers D18S68 and D18S1161. Two other chromosomal regions, 5q15–21 (between D5S644 and D5S2027) and 13q33-qter (between D13S1265 and 13qter), exhibited suggestive evidence of linkage (LOD > 2.0). Further, two previously reported linkage peaks on 9q34 and 17q24 were replicated in family specific analyses. No significant X chromosome linkage peaks were identified. In this genome-wide scan we demonstrate for the first time, that BAV and/or associated CVM exhibit linkage to chromosomes 18q, 5q and 13q. These regions likely contain genes whose mutation results in BAV and/or associated CVM indicating their important role in valvulogenesis and cardiac development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lisa J. Martin and Vijaya Ramachandran have contributed equally to this work.     

Mouse Af9 is a controller of embryo patterning,like Mll,whose human homologue fuses with Af9 after chromosomal translocation in leukemia

Chromosomal translocation t(9;11)(p22;q23) in acute myeloid leukemia fuses the MLL and AF9 genes. We have inactivated the murine homologue of AF9 to elucidate its normal role. No effect on hematopoiesis was observed in mice with a null mutation of Af9. However, an Af9 null mutation caused perinatal lethality, and homozygous mice exhibited anomalies of the axial skeleton. Both the cervical and thoracic regions were affected by anterior homeotic transformation. Strikingly, mice lacking functional Af9 exhibited a grossly deformed atlas and an extra cervical vertebra. To determine the molecular mediators of this phenotype, analysis of Hox gene expression by in situ hybridization showed that Af9 null embryos have posterior changes in Hoxd4 gene expression. We conclude that the Af9 gene is required for normal embryogenesis in mice by controlling pattern formation, apparently via control of Hox gene regulation. This is analogous to the role of Mll, the murine homolog of human MLL, to which the Af9 gene fuses in acute myeloid leukemias.     

Population-genetic basis of haplotype blocks in the 5q31 region

We investigated patterns of nucleotide variation in the 5q31 region identified by Daly et al. as containing haplotype blocks, to determine whether the blocklike pattern requires the assumption of hotspots in recombination. Using extensive simulations that generate data matched to the Daly et al. data set in (a) the method of ascertainment of single-nucleotide polymorphisms, (b) the heterozygosity of ascertained markers, (c) the number of block boundaries, and (d) the diversity of haplotypes within blocks, we show that the patterns found in the Daly et al. data are not consistent with the assumption of uniform recombination in a population of constant size but are consistent either with the presence of hotspots in a population of constant size or with the absence of hotspots if there was a period of rapid population growth. We further show that estimates of local recombination rate can distinguish between population growth and hotspots as the primary cause of a blocklike pattern. Estimates of local recombination rates for the Daly et al. data do not indicate the presence of recombination hotspots.     

AML1, the target of chromosomal rearrangements in human leukemia, regulates the expression of human complement receptor type 1 (CR1) gene.

The human CR1 gene is expressed specifically in hematopoietic cells. It is suggested that some cell-type specific factors which involve in gene-specific activation or repression exist in cells according to the result that the gene expression varies differently depend on differentiation stage. Here, we demonstrate that the integrity of a polyomavirus enhancer core sequence, 5'-TGTGGT-3', is critical to the human CR1 promoter activity. AML1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. We show that the AML1 binds specifically to this site and activates the human CR1 promoter. Furthermore, we demonstrate that the Ets binding site (GGAA) located 2 bp upstream of the AML1 site is also involved in the regulation of the human CR1 promoter activity. Point mutations of either the AML1 or the Ets binding site that abolish the binding of the respective factors result in significant decreases of the human CR1 promoter activity. These results suggest that AML1 and Ets proteins direct the expression of the human CR1 promoter.     

Cytogenetics of chronic T cell leukemia, including two patients with a 14q+ translocation.

Chromosome studies were done on 7 patients with chronic T cell leukemia. Their lymphocytes responded in culture to one or more T cell mitogens: PHA, Con A, or the calcium ionophore A23187. Clones of cytogenetically-abnormal cells were present in all seven patients, but on occasion the frequency of such cells varied greatly in cultures stimulated with different mitogens. There was no consistent chromosome change, but alterations of chromosome 2 were noted in four individuals and of chromosome 14 in three. In two patients, there was a translocation to the long arm of chromosome 14, producing a 14q+, with the break point in the terminal portion, an abnormality previously observed in B cell lymphomas. One of these patients also showed evidence of clonal evolution in sequential cytogenetic studies, but more data are needed to determine whether such investigations are of prognostic value with respect to the clinical course of the disease.     

Mapping of a new quantitative trait locus for resistance to malaria in mice by a comparative mapping approach with human Chromosome 5q31-q33

A number of linkage studies in human populations have identified a locus (pfbi) on Chromosome 5q31-q33 controlling Plasmodiun falciparum blood infection levels. This region contains numerous candidate genes encoding immunological molecules such as cytokines, growth factors and growth-factor receptors. We have used an F₁₁ advance intercross line (AIL) population of mice infected with Plasmodium chabaudi to identify additional mouse quantitative trait loci (QTL) for control of parasitaemia on Chrs 11 and 18, which carry regions homologous to human Chr 5q31-q33. Herein, we report a novel QTL for parasitaemia control (char8) on the mouse Chr 11, linked to marker D11Mit242, and involved in the clearance stages of the parasites from the bloodstream. Strikingly, several Th2 cytokines that are located within char8 have been identified to play a predominant role in the late stages of the infection.     

Infertility in mice infected genitally with a human strain of Chlamydia trachomatis

Progesterone-treated C3H and TO mice were inoculated genitally with a human C. trachomatis strain, serovar E, designated N.I. 1 or with 2SP control medium. Of the C3H mice serving as controls 93% had litters by the end of a 6-month period compared to 31% of mice infected with chlamydiae. This infertility could not be explained by tubal occlusion, since the oviducts appeared normal at autopsy. Some of the mice were induced to superovulate. Eggs were never recovered from the oviducts on the inoculated sides of infertile mice although they were sometimes found in the lumen of the uninoculated oviducts. In contrast, eggs were recovered routinely from both oviducts of control mice. In addition, eggs and/or their accompanying cumulus cells could be seen in the periovarial space of mice inoculated with chlamydiae, indicating a failure of the transportation of eggs to the oviduct. This could explain the high incidence of ectopic pregnancies in women after chlamydial infection. No adverse effect on fertility was seen in TO mice inoculated genitally with strain N.I.1. Of the mice given 2SP medium, 73% had litters, but 87% of the mice inoculated with chlamydiae were also fertile. There was, however, a significantly greater variation in the birth weights of mice born to infected TO mothers than those born to control mice. This difference in the susceptibility of mouse strains suggests that a genetic predisposition should also be considered for man.