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PubMed1995

Naturally processed peptides from rheumatoid arthritis associated and non-associated HLA-DR alleles.

Naturally processed peptides from immunoaffinity-purified HLA-DRB1*0401, -DRB1*0404 (rheumatoid arthritis (RA)-associated), and -DRB1*0402 (non-RA-associated) molecules were analyzed by capillary liquid chromatography and mass spectrometry. The molecular weights observed for more than 60 eluted peptides from each HLA-DR protein ranged from 788 to 3535 atomic mass units, corresponding to peptides 7 to 32 amino acids in length. Sequencing of more than 60 of the abundant peptides revealed nested sets of peptides that were derived from only 12 different proteins. The majority of these proteins were membrane-associated (HLA class I, class II, and Ig molecules). Synthetic peptides, corresponding to endogenous peptide sequences, bound with high affinity (5 to 80 nM) to the HLA-DR molecules from which they were eluted. In addition, most were promiscuous binding peptides in that they also bound to other HLA-DR molecules. Truncations of eluted peptide sequences and alanine scanning mutational analysis of a Mycobacterium leprae peptide were used to identify the peptide residues involved in binding to DRB1*0404 and DRB1*0402 molecules. Furthermore, an invariant chain peptide was eluted from the DRB1*0402 molecules but not from the RA-associated molecules. The lack of invariant chain peptides from DRB1*0401 and DRB1*0404 molecules may contribute to the loading of autoantigen peptides into these molecules and to their association with disease.

Alleles↗
PubMed1995

Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain.

The beta-amyloid precursor protein (APP) is proteolytically processed to generate beta-amyloid protein, the principal protein component of neuropathological lesions characteristic of Alzheimer's disease. To investigate potential sorting signals in the cytoplasmic tail of APP, we transplanted APP cytoplasmic tail sequences into the cytoplasmic tail of the human transferrin receptor (TR) and showed that two sequence motifs from the APP cytoplasmic tail promote TR internalization. One sequence, GYENPTY, is related to the low density lipoprotein receptor internalization signal, FDNPVY, but also involves a critical glycine residue; the other, YTSI, conforms to the 4-residue tyrosine-based internalization signal consensus sequence. Furthermore, a chimeric molecule (APP-TR) consisting of the cytoplasmic domain of APP and the transmembrane and external domains of TR was rapidly internalized enabling the transport of iron into the cell at approximately 50% the rate of wild-type TR. Alanine scanning mutations indicated that the two sequences identified in transplantation experiments were required for internalization of the chimera. Metabolic pulse-chase experiments showed that the APP-TR chimeras were degraded in a post-Golgi membrane compartment within 2-4 h following normal glycosylation. Degradation was partially dependent upon the two internalization signals and was inhibited by ammonium chloride. A fraction of APP-TR chimeras traffic to a degradative endocytic compartment after appearing on the cell surface. Comparison of soluble APP released from cells expressing either full-length human APP or mutant APP with the sequence YENPTY deleted indicated that this sequence is required for sorting of full-length APP along similar trafficking pathways as the APP-TR chimera.

Amino Acid Sequence↗
PubMed1994

Characterization of the regulatory elements in the promoter of the human elongation factor-1 alpha gene.

Most eukaryotic cells abundantly express polypeptide chain elongation factor-1 alpha (EF-1 alpha), an enzyme which catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes. In this study, a series of deletion and scanning mutations was introduced in the promoter region of human EF-1 alpha chromosomal gene. Mutated promoters were fused to the bacterial chloramphenicol acetyltransferase gene, and their promoter activity was determined in human HeLa cells. These analyses indicated that both the 5'-flanking region and the first intron of the EF-1 alpha gene are essential for its promoter activity. The region responsible in the intron contains several Sp1 and Ap1 elements which seem to have additive effects on its promoter activity. In the 5'-flanking region, two cis-elements (EFP1 and EFP2) which work interdependently were identified. Gel shift assay with EFP1 and EFP2 elements indicated that several nuclear factors bind to EFP1 and EFP2, and one of the three retarded bands with EFP2 could be super-shifted with the anti-Sp1 antibody. These results indicate that Sp1 or its related factor cooperatively enhances the expression of the EF-1 alpha gene in the 5' flanking region.

Base Sequence↗
PubMed1991

Direct molecular identification of the mouse pink-eyed unstable mutation by genome scanning.

DNA sequences associated with the mouse pink-eyed unstable mutation were identified in the absence of closely linked molecular markers and without prior knowledge of the encoded gene product. This was accomplished by "genome scanning," a technique in which high-resolution Southern blots of genomic DNAs were hybridized to a dispersed and moderately repetitive DNA sequence. In this assay, pink-eyed unstable DNA was distinguished from the DNA of wild-type and revertant mice by enhanced hybridization to one of several hundred resolved fragments. The fragment showing enhanced hybridization in pink-eyed unstable DNA was cloned and found to lie within a DNA duplication that is located close to, or within, the pink-eyed dilution locus. The duplication associated with the mouse pink-eyed unstable mutation may mediate the high reversion frequency characteristic of this mutation.

Alleles↗
PubMed2007

Prevalence and functionality of paucimorphic and private MC4R mutations in a large, unselected European British population, scanned by meltMADGE.

Identification of unknown mutations has remained laborious, expensive, and only viable for studies of selected cases. Population-based "reference ranges" of rarer sequence diversity are not available. However, the research and diagnostic interpretation of sequence variants depends on such information. Additionally, this is the only way to determine prevalence of severe, moderate, and silent mutations and is also relevant to the development of screening programs. We previously described a system, meltMADGE, suitable for mutation scanning at the population level. Here we describe its application to a population-based study of MC4R (melanocortin 4 receptor) mutations, which are associated with obesity. We developed nine assays representing MC4R and examined a population sample of 1,100 subjects. Two "paucimorphisms" were identified (c.307G>A/p.Val103Ile in 27 subjects and c.-178A>C in 22 subjects). Neither exhibited any anthropometric effects, whereas there would have been >90% power to detect a body mass index (BMI) effect of 0.5 kg/m(2) at P=0.01. Two "private" variants were also identified. c.335C>T/p.Thr112Met has been previously described and appears to be silent. A novel variant, c.260C>A/p.Ala87Asp, was observed in a subject with a BMI of 31.5 kg/m(2) (i.e., clinically obese) but not on direct assay of a further 3,525 subjects. This mutation was predicted to be deleterious and analysis using a cyclic AMP (cAMP) responsive luciferase reporter assay showed substantial loss of function of the mutant receptor. This population-based mutation scan of MC4R suggests that there is no severe MC4R mutation with high prevalence in the United Kingdom, but that obesity-causing MC4R mutation at 1 in 1,100 might represent one of the commonest autosomal dominant disorders in man.

Aged↗
PubMed1990

Analysis of the mouse Dhfr/Rep-3 major promoter region by using linker-scanning and internal deletion mutations and DNase I footprinting.

The mouse dihydrofolate reductase (Dhfr) promoter region is buried within a CpG island (a region rich in unmethylated CpG dinucleotides), has a high G+C content, and lacks CAAT and TATA elements. The region contains four 48-bp repeats, each of which contains an Sp1-binding site. Another gene, Rep-3 (formerly designated Rep-1), shares the same general promoter region with Dhfr, being transcribed in the direction opposite that of Dhfr. Both genes appear to be housekeeping genes and are expressed at relatively low levels in all tissues. The 5' termini of the major Dhfr transcripts are separated from the 5' termini of the Rep-3 transcripts by approximately 140 bp. This curious structural arrangement suggested that the two genes might share common regulatory elements. To investigate the promoter sequences driving bidirectional transcription, a series of promoter mutations was constructed. These mutations were assayed by a replicating minigene system and by promoter fusions to the chloramphenicol acetyltransferase gene. Linker-scanning mutations that spanned the four repeats produced a variety of mRNA transcript phenotypes. The effects were primarily quantitative, generally reducing the abundance of transcripts for one or both genes. Some mutations affected Dhfr in a qualitative manner, such as by changing the startpoint of one of the major Dhfr transcripts or changing the relative abundance of the two major Dhfr transcripts. Additionally, protein transcription factors that bind to sequences in the mouse Dhfr/Rep-3 major promoter region, potentially affecting expression of either or both genes, were investigated by DNase I footprinting. The results indicate that multiple protein-DNA interactions occur in this region, reflecting potentially complex transcriptional control mechanisms that might modulate expression of either or both genes under different physiological conditions.

Animals↗
PubMed2006

Rapid mutation screening for HRPT2 and MEN1 mutations associated with familial and sporadic primary hyperparathyroidism.

Familial hyperparathyroidism, a disease of the parathyroid glands, may occur in conjunction with pituitary and pancreatic tumors (multiple endocrine neoplasia type I), kidney and bone tumors (hyperparathyroidism jaw tumor syndrome), or alone (familial isolated hyperparathyroidism). This study describes the development and validation of rapid scanning for mutations in two tumor suppressor genes linked to familial hyperparathyroidism-MEN1 and HRPT2. Denaturing high-performance liquid chromatography mutation scanning for MEN1 was performed using a set of 10 amplicons covering the nine coding exons and flanking intronic regions and for HRPT2 using a set of three amplicons for exons 1, 2, and 7 and flanking intronic regions, in which 80% of the mutations identified to date are located. All 52 MEN1 mutations or polymorphisms, 46 known and six unknown, were successfully detected. Mutation detection in exon 9 was not confounded by the presence of the common polymorphism D418D. In addition, all 10 HRPT2 mutations were successfully detected, and a two-step approach was able to distinguish IVS2 common polymorphisms from exon 2 mutations. The development of rapid denaturing high performance liquid chromatography mutation scanning of MEN1 and HRPT2 facilitates a molecular diagnosis of the associated familial syndromes for both clinically affected and at-risk family members.

Chromatography, Liquid↗
PubMed2003

Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas.

Neurofibromatosis 2 (NF2) is a severe autosomal dominant disorder that predisposes to multiple tumours of the nervous system. About half of all patients are founders with clinically unaffected parents. The purpose of the present study was to examine the extent to which mosaicism is present in NF2 founders. A total of 233 NF2 founders with bilateral vestibular schwannomas (BVS) were screened by exon scanning. NF2 mutations were detected in the blood samples of 122 patients (52%). In 10 of the 122 cases, the ratio of mutant to normal alleles was obviously less than 1, suggesting mosaicism. Tumour specimens were available from 35 of the 111 subjects in whom no mutation could be detected in blood specimens. Mutational analysis by exon scanning detected typical NF2 mutations in 21 of the 35 tumours. In nine subjects, the alterations found in tumours could be confirmed to be the constitutional mutation based on finding of identical mutations in pathologically and/or anatomically distinct second tumours. In six other subjects with only a single tumour available, allelic loss of the NF2 gene was found in addition to the mutation in each tumour, suggesting that either the mutation or the deletion of the NF2 gene is probably the constitutional genetic alteration. Our results suggest that failure to find constitutional mutations in blood specimen from these 15 patients was not because of the limitation of the applied screening technique, but the lack of the mutations in their leucocytes, best explained by mosaicism. Extrapolating the rate (15/35 = 43%) of mosaicism in these 35 cases to the 111 NF2 founders with no constitutional NF2 mutations found in their blood, we inferred 48 mosaic subjects (111 x 0.429). Adding the 10 mosaic cases detected directly in blood specimens, we estimate the rate of mosaicism to be 24.8% (58/233) in our cohort of 233 NF2 founders with bilateral vestibular schwannomas.

Base Sequence↗
PubMed2003

Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome.

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10,000 and 5/100,000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 x 10(-4). Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10(-6) using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (approximately 100 bp) juxtaposed with a 'clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent approximately 9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 x 10(-5) in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 x 10(4) turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10(5) persons.

Cell Line↗
PubMed1999

Multiplex analysis of mutations in four genes using fluorescence scanning technology.

Multiplex analysis of genetic mutations using fluorescence scanning methodology is an accurate, efficient, and cost-effective approach to genotypic characterization. Fluorescence labeling during the synthesis of polymerase chain reaction primers allows the application of this technology to well-established protocols. We have simultaneously analyzed the four polymorphisms of factor V Leiden (G1691A), prothrombin G20210A, 5,10-methylenetetrahydrofolate reductase C677T, and cystathionine beta-synthase 844ins68. Three of these mutations have been associated with an increased risk of thrombosis. Following polymerase chain reaction with fluorescence-labeled primers, the polymerase chain reaction products were digested with an appropriate restriction enzyme (if necessary for detection of the mutation), diluted into one tube per sample for co-loading (multiplex loading), and analyzed with GeneScan software for fragment analysis following capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Foster City, CA, USA). Multiplex loading increased throughput without compromising precision.

Cystathionine beta-Synthase↗
PubMed2004

High sensitivity scanning of colorectal tumors and matched plasma DNA for mutations in APC, TP53, K-RAS, and BRAF genes with a novel DHPLC fluorescence detection platform.

Tumor-associated DNA has been detected in plasma of colorectal cancer (CRC) patients using various techniques but with limited gene or mutation coverage. We report a highly sensitive scanning methodology for mutational assessment of the APC and TP53 genes, which typically pose an analytical challenge because of their significant genotypic heterogeneity as well as specific mutational scoring assays for K-RAS and BRAF. Plasma DNA isolated from 20 CRC patients were scanned for mutations in these targets without knowledge of the molecular or pathological analyses of the matched primary tumors. We chose mutation scanning technology and these molecular targets to provide a comprehensive screen for somatic mutations known to be associated with sporadic CRC. Mutations were identified with a novel denaturing high-performance liquid chromatography (DHPLC) platform that uses post-separation fluorescence technology to enable the detection of variants that represent <0.1% of the total analyzed DNA. Mutant allele specific amplification (MASA) followed by detection with the same platform was used to identify low-level target mutations (mutation scoring) in K-RAS codons 12, 13, and 61, and BRAF codon 599. Using this combined scanning and scoring approach, we were able to identify at least one mutational event in 20/20 (100%) CRC patients. The thoroughness of a mutation scanning and scoring panel may have important implications for CRC screening and disease monitoring during and following therapy.

Alleles↗
PubMed2004

Use of TP53 reference materials to validate mutations in clinical tissue specimens by single-strand conformational polymorphism analysis.

BACKGROUND: As genetic information moves from basic research laboratories in to the clinical testing environment, there is a critical need for reliable reference materials for the quality assurance of genetic tests. A panel of 12 plasmid clones containing wild-type or point mutations within exons 5-9 have been developed as reference materials for the detection of TP53 mutations. AIM: The goal of this study was to validate the reference materials in providing quality assurance for the detection of TP53 mutations in clinical specimens. METHODS: We studied 33 gynecological samples, 11 apparently normal samples and 22 malignant tumors of various origins. Mutations were identified using single-strand conformational polymorphism analysis with both slab gel and capillary electrophoresis. All DNA samples were amplified with fluorescently labeled PCR primers specific for exons 5-9 for mutation detection. RESULTS: Of the 33 patient samples tested, mutations and polymorphisms were found in six specimens in three of the five exons scanned; no mutations were found in exons 7 or 9. Both a mutation and polymorphism were found in non-malignant specimens from the control group. The mutations were confirmed by DNA sequence analysis of the regions scanned. CONCLUSIONS: Mutations and polymorphisms were detected in the clinical samples. All of the mutations were silent except for one non-conservative mutation in exon 5, codon 181. This study demonstrates the usefulness of the National Institute of Standards and Technology (NIST) TP53 reference panel in TP53 mutation detection in clinical tissue specimens.

Adolescent↗
PubMed2004

Scanning for MODY5 gene mutations in Chinese early onset or multiple affected diabetes pedigrees.

Mutation of HNF-1beta gene has been reported in early onset diabetes or MODY families and this gene has been defined as MODY5 gene. The aim of our study was to examine whether HNF-1beta mutation contribute to early onset or multiple affected diabetes pedigrees in Chinese. Molecular scanning of HNF-1beta gene promoter region, nine exons and flanking introns was performed in 154 unrelated probands from early onset and multiple affected diabetes Chinese pedigrees. The family members of probands with mutations or variants and 58 nondiabetics were also examined. Clinical examinations of renal morphology, renal function and beta-cell function were performed in the HNF-1beta gene mutation carriers and family members. Mutation of HNF-1beta gene causing the substitution S36F was found in two subjects of an early onset diabetic family. One carrier has early onset diabetes, renal function impairment and renal cyst, while the other has impaired glucose tolerance only. This is the first case of MODY5 gene mutation diabetes found in the Chinese. Three HNF-1beta variants were identified and no significant differences in allele frequencies for these variants were detected between the nondiabetic and diabetic groups. Nucleotide 66 of intron 8 of HNF-1beta gene was G in the Chinese population rather than A as noted in the GenBank sequence. These results suggest that HNF-1beta gene mutations may be associated with nondiabetic renal dysfunction and diabetes in Chinese, but they are responsible for only a small percentage of early onset or multiple affected diabetes pedigrees including MODY.

Adenine↗
PubMed2005

Detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis using denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.

Antibiotics, Antitubercular↗
PubMed1999

Rapid DNA mutation identification and fingerprinting using base excision sequence scanning.

Base excision sequence scanning (BESS) is a new polymerase chain reaction (PCR)-based mutation scanning method that locates and identifies all DNA mutations. The BESS method consists of two procedures that generate "T" (BESS T-Scan) and "G" ladders (BESS G-Tracker) analogous to T and G ladders of dideoxy sequencing. The BESS procedures are simple to perform and require no special equipment or gels, no reaction optimization beyond PCR, and no heteroduplex formation. The samples are analyzed on standard sequencing gels or on automated DNA sequencers, and the data produced are easy to interpret, requiring a simple comparison of the sequence of normal and mutant DNA. The BESS method is versatile, having applications not only for mutation detection, but also single nucleotide polymorphism (SNP) discovery and analysis, DNA fingerprinting (including viral and bacterial typing), and clone identification. In this study, we utilize BESS in two of these applications: detection of a point mutation in BRCA1, and DNA typing of human papilloma virus (HPV).

BRCA1 Protein↗