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At least 523 records · Page 29Linked to original sources
PubMed2006

Microarray-based mutation analysis of the ABCA4 gene in Spanish patients with Stargardt disease: evidence of a prevalent mutated allele.

PURPOSE: To evaluate, in a pool of affected families, the mutation spectrum in Stargardt patients from Spain, using the ABCR400 microarray that contains described sequence variants in the gene encoding for the photoreceptor specific ATP-binding cassette transporter (ABCA4). METHODS: We analyzed 76 Spanish patients with STGD1 for a population-specific survey on the sequence variations in the ABCA4 gene, using the ABCR400 microarray. RESULTS: Potential disease-associated alleles were identified in 91 of the 152 STGD1 chromosomes studied, resulting in a detection rate of 60%. The two mutant alleles were found in 33/76 patients (43%), whereas in 25/76 cases (33%) only one allele could be identified. In the remaining 18 patients no mutations were found. In total, we identified 40 sequence variations that could be related to the disease. The vast majority of these substitutions (35/40) were missense mutations. Three frameshift mutations and two splicing variants were also found. CONCLUSIONS: We identified a major disease-associated allele, R1129L, which accounted for 24% of the mutated alleles detected, and a high frequency (12%) of complex alleles.

ATP-Binding Cassette Transporters↗
PubMed1996

Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1.

The 2.6 Angstrom crystal structure for human cyclin-dependent kinase 2(CDK2) in complex with CksHs1, a human homolog of essential yeast cell cycle-regulatory proteins suc1 and Cks1, reveals that CksHs1 binds via all four beta strands to the kinase C-terminal lobe. This interface is biologically critical, based upon mutational analysis, but far from the CDK2 N-terminal lobe, cyclin, and regulatory phosphorylation sites. CDK2 binds the Cks single domain conformation and interacts with conserved hydrophobic residues plus His-60 and Glu-63 in their closed beta-hinge motif conformation. The beta hinge opening to form the Cks beta-interchanged dimer sterically precludes CDK2 binding, providing a possible mechanism regulating CDK2-Cks interactions. One face of the complex exposes the sequence-conserved phosphate-binding region on Cks and the ATP-binding site on CDK2, suggesting that CKs may target CDK2 to other phosphoproteins during the cell cycle.

Base Sequence↗
PubMed1996

Identification of the primary metal ion-activation sites of the diphtheria tox repressor by X-ray crystallography and site-directed mutational analysis.

The diphtheria tox repressor, DtxR, is a 226 amino acid transition metal ion-activated regulatory protein that controls the expression of diphtheria toxin in toxigenic Corynebacterium diphtheriae. The previously solved three-dimensional DtxR structures have identified two potential metal ion binding sites which may play a role in the activation of DNA binding by the repressor. We have used both X-ray crystallographic and site-directed mutational analysis of DtxR(C102D)-Ni2+ complexes and DtxR to identify the metal ion-binding site which results in the activation of the repressor. We demonstrate that DtxR contains both a primary and an ancillary metal ion binding site. The primary site functions directly in the activation of DNA binding. In contrast, the ancillary site contributes weakly, if at all, to activation.

Bacterial Proteins↗
PubMed2003

Mutation analysis of candidate genes in melanoma-prone families: evidence of different pathogenetic mechanisms at chromosome 9P21.

Putative tumour suppressor genes CDKN2A and CDKN2B (on chromosome 9p21) and CDKN2A-interacting cell growth regulatory genes CDK4 and Id-1 have been demonstrated to be involved in the pathogenesis of malignant melanoma (MM). Mutation analysis of these candidate genes was performed in MM families from southern Italy with three or more affected members or two affected members and one or more relative with histologically diagnosed atypical naevus. Two CDKN2A mutations, Arg24Pro and 1-292 G>A, were observed in two (15%) families; except for CDKN2A and Id-1 polymorphisms, no sequence variations were detected in the remaining genes. Screening among 119 sporadic MM cases revealed two additional CDKN2A mutations at very low prevalences. Identification of a large shared haplotype at 9p21 in some MM families negative for CDKN germline mutations suggests that other CDKN-inactivating mechanisms may be responsible for MM predisposition or, alternatively, additional susceptibility gene(s) may be present on chromosome 9p21. Fluorescence in situ hybridization analysis of a subset of MM tissue sections seemed to indicate that the D9S171 locus may be involved in MM pathogenesis.

Chromosomes, Human, Pair 9↗
PubMed2002

Mutational analysis of baculovirus phosphatase identifies structural residues important for triphosphatase activity in vitro and in vivo.

Baculovirus phosphatase (BVP) and mammalian capping enzyme (Mce1) are members of the RNA triphosphatase branch of the cysteine phosphatase superfamily. Although RNA triphosphatases have a core alpha/beta fold similar to other cysteine phosphatases, there is little conservation of primary structure outside of the cysteine-containing P-loop motif, HCxxxxxR(S/T), that comprises the active site. However, there is extensive primary structure conservation between members of the RNA triphosphatase branch, whether from cellular or viral sources and whether they are bifunctional capping enzymes such as Mce1 or monofunctional RNA phosphatases such as BVP. To evaluate the functional significance of such sequence conservation, we performed a mutational analysis of 14 residues of BVP. We identified three side chains (Trp6, Lys25, and Arg153) as essential for triphosphatase activity in vitro, i.e., W6A, K25A, and R153A were <0.1% as active as wild-type BVP, and were unable to complement a yeast RNA triphosphatase null mutant in vivo. Six other BVP residues (Thr62, Tyr67, Tyr68, Lys82, Glu158, and Arg159) were deemed functionally important, i.e., Ala mutations reduced triphosphatase activity to <20% of wild-type. On the basis of the locations of the equivalent amino acids in the Mce1 crystal structure, we surmise that the essential/important BVP residues ensure proper conformation of the catalytic P-loop (e.g., Arg153 and Tyr68) or other elements of the tertiary structure. Our results highlight a conserved Trp6-Lys25 pi-cation pair essential for BVP function.

Acid Anhydride Hydrolases↗
PubMedDate not supplied

Genomic organization and mutational analysis of the human UCP2 gene, a prime candidate gene for human obesity.

Uncoupling proteins (UCPs) are mitochondrial membrane transporters which are involved in dissipating the proton electrochemical gradient thereby releasing stored energy as heat. This implies a major role of UCPs in energy metabolism and thermogenesis which when deregulated are key risk factors for the development of obesity and other eating disorders. Recent studies have shown that the sympathetic nervous system, via norepinephrine (beta-adrenoceptors) and cAMP, as well as thyroid hormones and PPAR gamma ligands seem to be major regulators of UCP expression. From the three different UCPs identified so far by gene cloning UCP1 is expressed exclusively in brown adipocytes while UCP2 is widely expressed. The third analogue, UCP3, is expressed predominantly in human skeletal muscle and was found to exist in a long and a short form. At the amino acid level UCP2 has about 59% homology to UCP1 while UCP3 is 73% identical to UCP2. Both UCP2 and UCP3 were mapped in close proximity (75-150 kb) to regions of human chromosome 11 (11q13) that have been linked to obesity and hyper-insulinaemia. Furthermore, there is strong evidence that UCP2, by virtue of its ubiquitous expression, may be important for determining basal metabolic rate. Based on the published full-length cDNA sequence we have deduced the genomic structure of the human UCP2 (hUCP2) gene by PCR and direct sequence analysis. The hUCP2 gene spans over 8.4 kb distributed on 8 exons. The localization of the exon/intron boundaries within the coding region matches precisely the one found in the human UCP1 gene and is almost conserved in the recently discovered UCP3 gene as well. However, the size of each of the introns in the hUCP2 gene differs from its UCP1 and UCP3 counterparts. It varies from 81 bp (intron 5) to about 3 kb (intron 2). The high degree of homology at the nucleotide level and the conservation of the exon/intron boundaries among the three UCP genes suggests that they may have evolved from a common ancestor or are the result from gene duplication events. Mutational analysis of the hUCP2 gene in a cohort of 25 children of caucasian origin (aged 7-13) characterized by low BMR values revealed a point mutation in exon 4 (C to T transition at position 164 of the corresponding cDNA resulting in the substitution of an alanine residue by a valine at codon 55) and an insertion polymorphism in exon 8. The insertion polymorphism consists of a 45 bp repeat located 150 bp downstream of the stop codon in the 3'-UTR. The allele frequencies were 0.61 and 0.39 for the alanine and valine encoded alleles, respectively, and 0.71 versus 0.29 for the insertion polymorphism. Expression studies of the wildtype and mutant forms of UCP2 should clarify the functional consequences these mutations may have on energy metabolism and body weight regulation. In addition, mapping of the promoter region and the identification of putative promoter regulatory sequences should give insight into the transcriptional regulation of UCP2 expression--in particular by anyone of the above mentioned factors--in vitro and in vivo.

Adolescent↗
PubMed1994

Inhibition of inositol phosphate second messenger formation by intracellular loop one of a human calcitonin receptor. Expression and mutational analysis of synthetic receptor genes.

Receptors for calcitonin (CTRs) have been cloned from several species, and two isoforms have been found to be expressed in human tissue. One human CTR isoform (hCTR-1) contains a 16-amino acid insertion in its first intracellular (I1) loop that is not present in porcine CTR (pCTR), rat CTR, or the other human CTR (hCTR-2). To facilitate the study of CTRs by mutational analysis, we have constructed synthetic hCTR-1 and hCTR-2 genes. Activation of hCTR-1 expressed transiently in COS-1 cells stimulates the formation of cAMP but not of inositol phosphates (IPs) whereas pCTR, a chimeric CTR in which the I1 loop of pCTR was substituted for the I1 loop of hCTR-1, and hCTR-2 stimulate cAMP and IP formation. A series of chimeric CTRs in which intracellular loops 1, 2, and 3 and the carboxyl tail of pCTR were substituted individually or in combination for those of hCTR-1 were constructed. All chimeras stimulated cAMP formation whereas chimeras containing the I1 loop of hCTR-1 with its 16-amino acid insertion were incapable of stimulating IP formation. There was no correlation between maximal stimulation of cAMP and IP formation by these CTRs. Thus, an inserted sequence in the I1 loop of hCTR-1 abolishes stimulation of the IP signal transduction pathway while allowing stimulation of the cAMP pathway.

Amino Acid Sequence↗
PubMed2006

Expression status of RUNX1/AML1 in normal gastric epithelium and its mutational analysis in microdissected gastric cancer cells.

Although Runt-related transcription factors RUNXs (RUNX1-3) have a high similarity in their structure, only RUNX3 is known to be involved in gastric carcinogenesis. First, we examined mRNA expression of these three RUNX genes in the gastric mucosa, and, finding only RUNX2 was not expressed there, we further investigated RUNX1 and RUNX3 expression in three regions including the pit, isthmus/neck, and gland regions of the human normal stomach and whether RUNX1 is involved in gastric carcinogenesis. The mRNA expression of RUNX1 and RUNX3 was examined by use of the three regions isolated by laser-captured microdissection (LCM) and by use of primary gastric cancer tissues. Furthermore, RUNX1 mutational analysis was performed in the cancer cells, which also were isolated from 44 paraffin-embedded gastric cancer tissues by LCM. RUNX1 was co-expressed with RUNX3 in the pit region, and has cell growth-inhibition activity similar to RUNX3. RUNX3 has been reported to be suppressed by DNA methylation in a subset of gastric cancers; however, the expression of RUNX1 mRNA was observed in all of the gastric cancer cell lines and gastric cancer tissues that we examined. No RUNX1 mutation was found in the 44 gastric cancer patients. Although RUNX1 is similar to RUNX3 in both the expression pattern in the stomach and its cell growth-inhibition activity, RUNX1 is not involved in most cases of gastric cancers. These results suggest that the transcriptional target genes are different between these two family genes.

Cell Differentiation↗
PubMed2005

Mutation analysis of the WFS1 gene in seven Danish Wolfram syndrome families; four new mutations identified.

Wolfram syndrome (WS) is a neuro-degenerative autosomal recessive (AR) disorder (OMIM #222300) caused by mutations in the WFS1 gene on 4p16.1. More than 120 mutations have been identified in WFS1 associated with AR WS, as well as autosomal dominant nonsyndromic low-frequency sensorineural hearing loss (LFSNHL). WFS1 variants were identified in eight subjects from seven families with WS, leading to the identification of four novel mutations, Q194X (nonsense), H313Y (missense), L313fsX360 (duplication frame shift) and F883fsX951 (deletion frame shift), and four previously reported mutations, A133T and L543R (missense), V415del (in frame triple deletion) and F883fsX950 (deletion frame shift). A mutation was found in 11/14 disease chromosomes, two subjects were homozygous for one mutation, one subject was compound heterozygous for two nucleotide substitutions (missense), one subject was compound heterozygous for a duplication and a deletion (frame shift), and in three families only one mutation was detected (Q194X and H313Y). All affected individuals shared clinically early-onset diabetes mellitus and progressive optic atrophy with onset in the first and second decades, respectively. In contrast, diabetes insipidus was present in two subjects only. Various degrees and types of hearing impairment were diagnosed in six individuals and cataract was observed in five subjects.

Adolescent↗
PubMed2004

A solitary Peutz-Jeghers-type hamartomatous polyp in the duodenum. A case report including results of mutation analysis.

BACKGROUND/AIMS: We report a case of solitary Peutz-Jeghers-type hamartomatous polyp of the duodenum in a 22-year-old Japanese woman along with the results of genomic analysis. METHODS/RESULTS: The patient was almost asymptomatic, though endoscopic examination revealed a solitary lobular polypoid lesion measuring 3 cm in diameter in the first portion of the duodenum. The lesion was resected endoscopically. Histopathological examination showed hyperplasia with a tree branch-like extension of the lamina propria derived from the muscularis mucosae, consistent with histological features of polyps of Peutz-Jeghers syndrome (PJS). No mucocutaneous pigmentation of the skin was evident and family history was negative. Analysis of the loss of heterozygosity at the locus of 19p 13.3 and mutation analysis of the STK11/LKB1 gene, which has recently been recognized as a susceptible gene in PJS, were performed. However, no evidence of genomic abnormality was found. CONCLUSION: The clinical and investigative findings in our case suggest that the solitary Peutz- Jeghers-type hamartomatous polyp can be regarded as a clinical entity separate from PJS.

Adult↗
PubMed1993

Mutational analysis of a signal sequence required for protein secretion in Leishmania major.

In eukaryotes, amino-terminal extensions, signal sequences, mediate the translocation of lysosomal, membrane and secreted proteins into the lumen of the endoplasmic reticulum (ER). Structure/function studies indicate that eukaryotic signal sequences are composed of 3 distinct domains, a positively charged amino-terminal domain, a central hydrophobic domain, and a polar carboxy-terminal domain. In an attempt to better understand protein trafficking in Leishmania we have constructed strains of Leishmania major that secrete an exogenous protein, interferon-gamma (IFN-gamma), under the control of a mammalian signal sequence. In this report we present a mutational analysis of this signal sequence. Deletion of the entire signal sequence or the hydrophobic core region prevents secretion of IFN-gamma and results in cytoplasmic expression of the protein. Mutations in the amino-terminal domain indicate that a net positive charge is not required for efficient secretion of IFN-gamma. Mutations in the carboxy-terminal domain are more complex and display two phenotypes, either they prevent expression of IFN-gamma or they have no effect on protein secretion. These results indicate that the function of the signal sequence in targeting proteins to the ER in Leishmania is similar to that observed in yeasts and higher eukaryotes and suggests that the Leishmania protein secretory apparatus may also be similar.

Amino Acid Sequence↗
PubMed2004

[Mutation analysis of ganglioside-induced differentiation associated protein-1 gene in Chinese Charcot-Marie-Tooth disease].

OBJECTIVE: To study the mutation feature of ganglioside-induced differentiation associated protein-1 (GDAP1) gene in Chinese Charcot-Marie-Tooth disease(CMT) patients. METHODS: Mutation analysis was carried out by use of polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) combined with DNA direct sequencing of the six exons and their flanking regions of GDAP1 gene in twenty-three CMT patients, including 8 probands of autosomal recessive CMT families and 15 sporadic patients. RESULTS: A compound heterozygous mutation A533G and A767G were unveiled in one autosomal recessive CMT kindred. The homozygous and heterozygous T507G were common SNPs in Chinese population. CONCLUSION: A533G and A767G of GDAP1 gene were new mutations firstly reported.

Adolescent↗
PubMed1998

Genomic organization and mutation analysis of p73 in oligodendrogliomas with chromosome 1 p-arm deletions.

p73, a protein having substantial structural and functional similarity to p53, has recently been identified and demonstrated to be a potential tumor suppressor. Its location on human chromosome 1p36.33 implicates p73 as a candidate for neuroblastoma. Like neuroblastoma, oligodendrogliomas also show a high frequency of deletions in chromosome 1p36.3. To determine whether p73 is a potential tumor suppressor gene involved in the development of oligodendrogliomas, we performed mutation analysis of p73 in oligodendrogliomas with chromosome 1 p-arm deletions. We first determined the genomic organization and the intron-exon boundary sequences of the p73 gene by long PCR, vectorette PCR, and Southern hybridization. This gene spans about 65 kb with a large first intron. Primer pairs for the amplification of each of the 13 p73 encoding exons were designed in corresponding introns. The amplicons were then analyzed using the denaturing high-performance liquid chromatography system for mutations in the p73 gene. Twenty oligodendroglioma samples with 1p36.3 deletions were screened, but no mutations were detected except for several polymorphisms. It is thus clear that p73 is not a candidate gene for oligodendroglioma despite its location in the frequently deleted 1p36.3 region.

Chromosomes, Human, Pair 1↗
PubMed2006

Myoepithelial carcinoma arising in an adenomyoepithelioma of the breast: case report with immunohistochemical and mutational analysis.

Adenomyoepithelioma (AME) of the breast is an uncommon tumor characterized by biphasic proliferation of both epithelial and myoepithelial cells. In rare instances, the epithelial, the myoepithelial or both components of an AME may become malignant. Described herein is the case of a 69-year-old woman who presented with myoepithelial carcinoma of the breast in an AME. Malignancy of myoepithelial component (MEC) was evidenced by the presence of cytological atypia, high mitotic rate, necrosis and local invasion. Immunohistochemical study demonstrated strong expression of P53 and phosphorylated extracellular signal-regulated kinase 1/2 in MEC. Laser capture microdissection technique and mutational analysis further revealed point mutation of the p53 gene (T-->G transversion at codon 270) in this population, but not in glandular epithelial cells or adjacent normal ductal epithelium. No mutations in exons 1 and 2 of the K-, H-, and N-ras genes were identified in any of the neoplastic component. To the authors' knowledge this is the first report of a mutation in the p53 gene in a malignant AME of the breast.

Aged↗
PubMed1993

Mutational analysis of hepatitis B virus enhancer 2.

Hepatitis B virus (HBV) infection is associated with the development of hepatocellular carcinoma in humans. HBV infection is primarily restricted to hepatocytes, and replication and gene expression of HBV require liver-specific transcription factors. Regulation of HBV gene expression has been shown to be controlled by two enhancers, and liver-specific gene expression of HBV can be attributed largely to the activity of enhancer 2. In this study, we have used mutational analysis to identify a 20-base-pair sequence motif essential for the liver-specific enhancer 2 activity. Analysis of the sequence reveals that this motif is similar to the regulatory region of several other liver-specific cellular genes, suggesting that common transcription factors may be involved in the activation of cellular as well as HBV gene expression in hepatocytes.

Base Sequence↗
PubMed1999

Mechanism of ligand recognition by BmrR, the multidrug-responding transcriptional regulator: mutational analysis of the ligand-binding site.

The Bacillus subtilis transcriptional regulator BmrR recognizes dissimilar hydrophobic cations and, in response, activates the expression of a multidrug transporter which expels them out of the cell. The structure of the inducer-binding domain of BmrR, both free and in complex with one of the inducers, tetraphenylphosphonium (TPP), revealed an unusual internal binding site, covered by an amphipathic alpha-helix. Upon unfolding of this helix, the TPP molecule penetrates into the core of the protein, where it contacts six hydrophobic residues and forms an electrostatic bond with a buried glutamate, E134 [Zheleznova et al. (1999) Cell 96, 353-362]. Here, a structure-based mutational analysis was used to understand how BmrR interacts with a wide variety of ligands. We determined the effects of alanine substitutions of each of the seven residues interacting with TPP, and mutations within the amphipathic alpha-helix, on the binding affinities of six different BmrR inducers. The E134A substitution abolished the binding of all but one inducer. Mutations of the hydrophobic residues contacting the ligand, and of the alpha-helix, had more moderate effects, often with the affinity for some inducers increasing and others decreasing as a result of the same substitution. These results indicate that each inducer forms a unique set of contacts within the binding site. The flexible geometry of this site and the lack of involvement of hydrogen bonds in ligand binding are the likely reasons for the extremely broad inducer specificity of BmrR. The similarly broad substrate specificity of multidrug transporters can be governed by the same structural principles.

Amino Acid Substitution↗
PubMed1999

Human transcription factor SLUG: mutation analysis in patients with neural tube defects and identification of a missense mutation (D119E) in the Slug subfamily-defining region.

Studies in mouse, chicken and Xenopus have shown that Slug is selectively expressed in the dorsal part of the developing neural tube. Ablation and antisense experiments in chicken suggest that Slug may be an important factor during neural tube closure. We therefore investigated the role of Slug as a possible candidate contributing to the aetiology of neural tube defects (NTD) in humans. We characterised the genomic structure of human SLUG including determination of the exon-intron boundaries. The coding sequence of SLUG was screened for mutations in 150 patients with NTD using single strand conformation analysis (SSCA). In one patient, we identified a missense mutation 1548C-->A in exon 2 causing an exchange of a conserved amino acid (D119E) in the Slug subfamily-defining region preceding the first zinc finger. This is the first description of a human mutation in the SLUG gene. In accordance with the findings in model organisms, the SLUG mutation may be causally related to the development of NTD in our patient and could be considered as a predisposing factor.

Adult↗
PubMed2000

Complete genomic organization of the human JAK3 gene and mutation analysis in severe combined immunodeficiency by single-strand conformation polymorphism.

JAK3 deficiency in humans results in autosomal recessive T-B+ severe combined immunodeficiency disease (SCID), a severe immunodeficiency that can only be cured by bone marrow transplantation. We unraveled the complete organization of the human JAK3 gene, which includes 23 exons. This information allowed us to set up a molecular screening test that enabled us to diagnose JAK3 deficiency in 14 patients from 12 unrelated families with T-B+ SCID. In order to define the mutations, we used a nonradioactive single-strand conformation polymorphism (SSCP)/heteroduplex (HD) assay based on exon-specific polymerase chain reaction (PCR). In this cohort of patients, 15 independent JAK3 gene mutations have been identified, including 7 that have not been described previously. Mutation analysis information was used for genetic counseling and prenatal diagnosis.

Consanguinity↗