Gene Function & Disease最新文献

Age-related macular degeneration (ARMD) is the leading cause of blindness in the elderly. A population-based segregation study as well as various twin studies suggest a role for genetic factors. Since ARMD shares some ocular phenotypic features with Best disease, we hypothesized that the Best disease gene may be involved in some cases of ARMD. In this report we have tested this possibility in two familial and two sporadic cases of ARMD. Our analysis failed to identify any disease-causing or polymorphic changes in the bestrophin gene. This, however, does not completely exclude the bestrophin gene as a candidate gene, because mutations in the distal promoter region as well as within the intron-branch site cannot be ruled out. The above results indicate that the structural and functional changes in the bestrophin gene are not the major factors associated with disease phenotype of ARMD in the families analyzed.

Best macular dystrophy is an autosomal dominant disease leading to macular degeneration and subsequent impaired vision. The disease has juvenile onset and affects the retinal pigment epithelium and adjacent photoreceptors. There are histopathological similarities between Best macular dystrophy (BMD) and age-related macular degeneration (AMD) with accumulation of lipofuscin in the outer retina. Recently, we identified the gene VMD2 causing Best macular dystrophy. The VMD2 gene has unknown function and there are no similarities between the VMD2 product, called bestrophin, and other proteins with known function. In order to gain more knowledge about the function of bestrophin we investigated its subcellular localization. DNA constructs encoding the bestrophin protein fused to the green fluorescent protein (GFP) or a c-myc tag were transiently expressed in COS-7 cells or retinal pigment epithelium cells. The observed pattern of bestrophin fusion protein was spotted and mainly perinuclear, well corresponding to the endoplasmic reticulum (ER), which was also suggested when counterstaining with an ER probe. Probes for other organelles had a different localization pattern compared to bestrophin. In conclusion, the results indicate that bestrophin is located to the endoplasmic reticulum.

Graves’ disease (GD) results from an interplay of genetic factors and environmental triggers. We examined the immunoregulatory gene, Bruton’s tyrosine kinase (BTK), a candidate gene located in a region on chromosome Xq21.33−22 which we have previously found to be linked to GD and designated as GD-3. The study aimed to identify whether BTK was GD-3. No association was found between GD and alleles of the marker DXS178 and the A/C SNP. We concluded that BTK is not the GD-3 Graves’ disease susceptibility gene on chromosome Xq21.33−22.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Furthermore, the involvement of this gene in other pathologies „associated with CF”︁, like Congenital Absence of the Vas Deferens (bilateral or unilateral, CBAVD or CUAVD, resp.) is known, though the mutations causing these phenotypes within a defined population are different from those observed in CF patients. While cystic fibrosis patients have CFTR mutations in both alleles, most of the patients suffering from CF-related pathologies have mutations in only one allele. Frequently, the second CFTR allele is not mutant but polyvariant carrying a combination of alleles of polymorphic loci, called Polyvariant Mutant Genes (PMG) which, as a whole haplotype, contribute to lowering the level of CFTR transcripts. The loci involved in these PMG are Tn, TGm (both in intron 8), and M470V (exon 10) of the CFTR gene. We have carried out an exhaustive analysis of the CFTR gene in order to determine its possible role in 15 azoospermic Spanish patients. We have found that all CBAVD cases with a CF mutation carry the haplotype 5T-12(TG)-V470 on the second allele which is different from that found in other related pathologies causing azoospermia, like Congenital Bilateral Absence of Seminal Vesicles (CBASV) or CUAVD, where we have found the haplotype 5T-11(TG)-M470 which is more efficient producing CFTR protein. We believe that the latter phenotypes represent incomplete forms of CBAVD. Given the high frequency of severe CF mutations an exhaustive screening of CFTR mutations should be offered when assisted reproduction technologies are available. Moreover, when two mutations (or one mutations and a PMG) are detected, their phase should be established, since the existence of double mutants could modify the phenotype and it may suggest that the alteration in the other CFTR gene remains unidentified.

A new protocol, termed methylation-sensitive amplicon subtraction (MS-AS), has been developed for the identification and cloning of aberrantly methylated DNA segments in complex genomes. This method is based on the preparation of amplicons from HpaII fragments and combines the normalization of relative DNA sequence abundancy and subtractive hybridization. The normalization step applies a specific form of PCR that permits the exponential amplification of differences in HpaII fragment representations, whereas amplification of common sequences is repressed. In contrast to other subtractive hybridization protocols for genomic DNA analyses, the MS-AS method requires only one cycle of competitive hybridization for the efficient enrichment of target molecules. MS-AS analyses of adenovirus type 12 (Ad12)-transformed or bacteriophage lambda DNA-transgenic hamster cell lines have led to the identification of several CpG-rich cellular gene fragments the methylation of which has been altered in the transgenic as compared to the non-transgenic cell lines. The new method adds to the growing number of genome scanning approaches, including methylation-sensitive representational difference analysis, restriction landmark genomic scanning, and methylation-sensitive arbitrarily primed PCR which all have been used to detect altered methylation sites in cancer cells.

The number of fully sequenced genomes available is rapidly increasing, and experiments are underway to get quantitative information of the transcription and expression of the different gene products. As this is progressing and projects on structural genomics are starting, the lack of readily accessible functional information about proteins is becoming more obvious. Here, we describe the comprehensive enzymatic and metabolic information system BRENDA (BRaunschweig ENzyme DAta base) which has been created - and is continuously updated - by manual extraction and evaluation of functional and molecular parameters on enzymes from the primary literature, presently containing parameters from approx. 35 000 literature references, holding data of at least 20 000 different enzymes from more than 4330 different organisms classified in the approx. 3 700 EC-numbers. More than 260 000 different enzyme/ligand relationships are given in the data repository. BRENDA is available via the world wide web (www.brenda.uni-koeln.de).

The absence of the FMR1 (fragile X mental retardation gene 1) gene product, protein FMRP (fragile X mental retardation protein) is causing the fragile X syndrome. FMRP, together with two homologues, called FXR1P and FXR2P, belongs to a small family of RNA-binding proteins (FXR proteins). The precise physiological function of the FXR proteins is unknown, but a role in mRNA transport has been suggested. In the present study, we have performed immunolocalization of these proteins during the embryonic development of the mouse to get more insight in their physiological function. All three proteins are expressed during mouse embryonic development, however, the pattern and intensity varies for each protein at the different developmental stages. During early development, the distribution of the Fxr proteins exhibits high similarities, however, during late development and in the neonate a more differential expression is observed especially in some non-neural tissues. The results of this descriptive study are discussed in relation to the pathogenesis of the fragile X syndrome.

Human myeloma cell lines are useful as an in vitro model for the genetic and cellular biological characterization of primary myeloma cells. We have been attempting to establish myeloma lines since 1979 for the study of the cytogenetic, molecular and cellular biological aspects of myeloma cells and to resolve the clinical problems associated with multiple myeloma. The establishment and characterization of some of these lines have been reported. Here we review the lines established at Kawasaki Medical School and discuss investigations related to the clinical manifestation of myeloma using these lines. For instance, the relation between myeloma specific chromosomal translocations, such as t(11;14)(q13;q32), t(8;14)(q24;q32), and t(4;14)(q16.3;q32.3), found in some of the cell lines and gene expression was examined.

In familial cases of mammary and ovarian carcinomas, particularly in those occurring at an early age, numerous mutations have been described in the human BRCA1 and BRCA2 genes located on chromosomes 17q21 and 13q12.3, respectively. We have identified a Caucasian family with several members suffering from mammary and/or ovarian carcinomas, most of them before the age of 50. The joint occurrence of several other carcinomas in this family, e.g., gastric carcinoma or hypernephromas, alerted our interest to study possible molecular causes. The index patient had breast cancer at age 26, bilateral cancer of the ovaries and Fallopian tubes at age 37, and hypernephroma at 46 years. From DNA of the index patient and her mother, we PCR-amplified and determined the nucleotide sequence of all 22 exons coding for the protein and parts of the flanking introns of the BRCA1 gene. Six polymorphic, probably non-consequential nucleotide exchanges were found in exons 11, 13, and 16. In both women we detected a 10 nucleotide pair insertion in exon 6 of the BRCA1 gene. This insertion led to the truncation of the gene product beyond amino acid 82. RT-PCR experiments using oligodeoxyribonucleotide primers located in exons 2 and 10 revealed biallelic expression of the BRCA1 gene in peripheral white blood cells (PWBCs) of the index patient and of normal individuals. The results of a protein truncation test performed with either mRNA from the index patient’s PWBCs or with subcloned cDNA fragments confirmed the biallelic expression of the normal and the truncated BRCA1 alleles. When studying transcription patterns of the BRCA1 gene, we found several splicing variants in the 5′-part of the gene involving exons 3, 5, 6, 7, the first codon of exon 8, and exons 9 and 10. In some of these alternate splice products, the RING finger motif encoded by exons 3 and 5 was obliterated. This observation was also made with mRNAs from PWBCs of healthy individuals or from different human cell lines. This alternate splicing pattern is not directly relevant in eliciting the oncogenic phenotype, but may contribute to a reduction in the amount of full length BRCA1 protein below a critical level.