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PubMed2006

Absence of TREM2 polymorphisms in patients with Alzheimer's disease and Frontotemporal Lobar Degeneration.

Triggering Receptor Expressed on Myeloid cells (TREM)2 deficiency originates a genetic syndrome characterized by bone cysts and presenile dementia, named Nasu-Hakola disease (NHD). Early onset dementia and marked involvement of frontal regions are features characterizing both NHD and other kinds of neurodegenerative disorders, such as Frontotemporal Lobar Degeneration (FTLD), and, in some cases, Alzheimer's disease (AD). Three Single Nucleotide Polymorphisms (SNPs) in TREM2 coding region were screened by allelic discrimination in a population of probable AD patients as well as FTLD patients as compared with age-matched controls. In addition, mutation scanning of the coding region of TREM2 gene was carried out in 7 patients with early onset AD (EOAD), 16 FTLD, and 20 controls. None of the SNPs analyzed was present, either in patients or controls. Moreover, mutation scanning of the five exons of TREM2 failed to detect the presence of novel polymorphisms. These data demonstrate that TREM2 coding region is highly conserved, implying a crucial role of this receptor. Further studies, including a functional analysis, are certainly required to clarify the role of TREM2 in neurodegenerative processes.

Aged↗
PubMed2006

A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting.

Rapid and reliable identification of deleterious changes in the breast cancer genes BRCA1 and BRCA2 has become one of the major issues in most DNA services laboratories. To rapidly detect all possible changes within the coding and splice site determining sequences of the breast cancer genes, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. All exons of both genes are covered by the DGGE scan, comprising 120 amplicons. We use a semiautomated approach, amplifying all individual amplicons with the same PCR program, after which the amplicons are pooled. DGGE is performed using three slightly different gel conditions. Validation was performed using DNA samples with known sequence variants in 107 of the 120 amplicons; all variants were detected. This DGGE mutation scanning, in combination with a PCR test for two Dutch founder deletions in BRCA1 was then applied in 431 families in which 52 deleterious changes and 70 unclassified variants were found. Fifteen unclassified variants were not reported before. The system was easily adopted by five other laboratories, where in another 3,593 families both exons 11 were analyzed by the protein truncation test (PTT) and the remaining exons by DGGE. In total, a deleterious change (nonsense, frameshift, splice-site mutation, or large deletion) was found in 661 families (16.4%), 462 in BRCA1 (11.5%), 197 in BRCA2 (4.9%), and in two index cases a deleterious change in both BRCA1 and BRCA2 was identified. Eleven deleterious changes in BRCA1 and 36 in BRCA2 had not been reported before. In conclusion, this DGGE mutation screening method for BRCA1 and BRCA2 is proven to be highly sensitive and is easy to adopt, which makes screening of large numbers of patients feasible. The results of screening of BRCA1 and BRCA2 in more than 4,000 families present a valuable overview of mutations in the Dutch population.

Ambulatory Care Facilities↗
PubMed1992

The GC box and TATA transcription control elements in the P38 promoter of the minute virus of mice are necessary and sufficient for transactivation by the nonstructural protein NS1.

To further define the transcriptional regulation of the P38 promoter in the minute virus of mice (MVM) genome, we constructed a series of internal deletion and linker scanning mutations. The mutant P38 constructs were assayed for transcriptional activity in vitro by primer extension analysis with nuclear extracts from murine A92L fibroblasts. Mutations which disrupted the GC box and TATA box severely reduced transcription in vitro. DNase I footprinting analysis confirmed that the murine transcription factor Sp1 bound to the GC box; however, no factors were observed interacting with a putative transcriptional activation regulatory element, termed the TAR element. The linker scanning mutations were analyzed in vivo by using a chloramphenicol acetyltransferase expression assay system, in both the presence and absence of constructs expressing the viral nonstructural protein, NS1. The ability of NS1 to transactivate the P38 promoter (up to 1,000-fold) depended entirely on the presence of intact GC and TATA box sequences. Disruption of the TAR element by either linker insertion mutations or an internal deletion did not inhibit transactivation of the P38 promoter. These results suggest that NS1 transactivates the P38 promoter indirectly by interacting with one or more components of the P38 core-transcription complex.

Animals↗
PubMed1989

Mutagenesis of the rat insulin II 5'-flanking region defines sequences important for expression in HIT cells.

To define the cis-acting elements important for rat insulin II gene expression, we analyzed the effects of 5' deletions and linker-scanning mutations on the expression of a rat insulin II reporter gene in an insulinoma cell line (HIT). The reporter gene contained 448 base pairs of 5'-flanking sequence joined to the bacterial chloramphenicol acetyltransferase gene. Expression of the 5' deletion mutations indicated that the minimal sequence requirement for efficient expression was 218 base pairs of 5'-flanking sequence, and at least three regions downstream from - 218 were important for transcription. A more precise localization of these elements and the cis-acting sequences in the promoter was achieved by analysis of the expression of 18 linker-scanning mutations. In these studies at least four other regions important for expression of the rat insulin II gene were identified. These findings suggest that the sequences important for rat insulin II and rat insulin I expression may differ significantly despite the high degree of sequence similarity in their 5'-flanking regions.

Animals↗
PubMed1989

Transcription of a poxvirus early gene is regulated both by a short promoter element and by a transcriptional termination signal controlling transcriptional interference.

The promoter region of an early gene (38K gene) of cowpox virus has been characterized by deletion and linker scanning mutational analyses. Modified versions of this promoter region were placed into the genome of vaccinia virus, and their transcriptional efficiencies were assessed by quantifying RNAs transcribed from these sequences. These analyses showed that the sequences in the region between 33 and 4 base pairs upstream of the transcriptional start site affect the efficiency of transcription from this promoter. Linker scanning mutations in the -27 to -10 region inhibited transcription. This region contains the sequence 5'-GAAAATATATT-3', which is present in at least two other early genes in the same positions (-21 to -11) relative to the transcriptional start sites of these genes. Elements of this sequence are similarly positioned in the promoter regions of several other poxvirus genes, suggesting that this sequence represents a transcriptional control element of at least a subset of poxvirus genes. The -8 to -2 sequence (5'-TTTTTAT-3') contains a transcriptional termination signal. Mutation of this sequence had two separate effects: (i) it reduced the efficiency of transcription from the promoter by approximately 30%, and (ii) it prevented this sequence from terminating the transcription from upstream genes. When overlapping transcription from upstream genes was not prevented by a termination signal present either within the 38K promoter or upstream of the promoter, transcription from this promoter was reduced by about 30%. This indicates that transcriptional termination has a role in the regulation of viral gene expression by controlling transcriptional interference.

Amino Acid Sequence↗
PubMed2000

Mutation analysis of the hamartin gene using denaturing high performance liquid chromatography.

Denaturing high performance liquid chromatography (DHPLC) is a novel high-capacity technique for gene mutation scanning. We have assessed the sensitivity and specificity of this method for analysis of the full coding sequence of the hamartin (TSC1) gene in 20 tuberous sclerosis patients, whose TSC1 genes previously had been studied by single strand conformation polymorphism analysis and protein truncation assay. All eight sequence variants previously identified were adequately detected by DHPLC. Additionally, this approach picked up three polymorphisms, one of which (IVS13-55 C>G) was hitherto unreported, therefore serving as proof of principle for this technique. Thus, DHPLC appears to be a highly sensitive method with advantages in terms of flexibility, fragments size analysis, cost and time and labor sparing, compared to classical approaches of mutation scanning.

Chromatography, High Pressure Liquid↗
PubMed1993

Characterization of the role of individual protein binding motifs within the hepatitis B virus enhancer I on X promoter activity using linker scanning mutagenesis.

A combination of linker scanning mutagenesis and deletional analyses has been used to determine the role of individual DNA-protein binding sites on expression from the hepatitis B virus (HBV) enhancer I-X promoter (map position (mp) 1042-1354, HBV adw2). Linker scanning mutation of the EF-C site caused a 67.5% drop in X promoter activity in HuH7 cells, but had no effect in HepG2 or HepSK cells. Mutation of the E element resulted in an approximately 50% reduction in X promoter activity in HuH7, HepG2, and HepSK cells. Deletional analysis showed that sequences upstream of the EF-C site (mp 1163) were required for full X promoter activity and implicated the NF-1a site as being sufficient for basal X promoter activity. However, PCR-directed linker scanning mutation of the NF-1a site did not cause a reduction in X promoter activity, indicating that this site was not an essential component of the X promoter. Taken together, these results indicated that multiple, partially redundant protein:DNA interactions in the enhancer I are essential for full X promoter activity. The lack of an essential basal promoter element supports the suggestion that the two separate HBV enhancer elements (enhI and enhII) were created by integration of the X gene into a primordial enhancer element.

Base Sequence↗
PubMed2004

Isolation and characterization of the canine serotonin receptor 1B gene (htr1B).

The serotonin receptor 1B gene (htr1B) has been suggested to be implicated in mental disorders in both humans and other species. We have isolated a canine bacterial artificial chromosome (BAC) clone containing htr1B, revealed the coding and surrounding DNA sequence of canine htr1B and designed primer sets for genomic sequencing of the gene. A mutation scan in 10 dogs revealed five single nucleotide polymorphisms in the htr1B coding sequence. By random sequencing of subclones of the BAC a polymorphic microsatellite repeat was found. We found evidence for at least four extended haplotypes in six dogs of the same breed. The chromosomal localization of the gene was confirmed by fluorescence in situ hybridisation and radiation hybrid mapping. This work provides a starting point for mutation scans and association studies on dogs with behavioural problems.

Amino Acid Sequence↗
PubMed2003

Identification of upstream sequences essential for activation of a bacteriophage P2 late promoter.

We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, P(F), which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the -35 region of the promoter. Introduction of a sigma(70) -35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor sigma(70) contacts and helps to recruit RNA polymerase holoenzyme.

Bacteriophage P2↗
PubMed1999

Activation of baculovirus very late promoters by interaction with very late factor 1.

Very late factor 1 (VLF-1) of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) activates the transcription of two genes, polyhedrin (polh) and p10, during the final, occlusion-specific phase of infection. Using transient expression assays responsive to VLF-1, we identified linker scan mutations in the polh and p10 promoters which abolished or weakened the ability of the promoters to respond to stimulation by VLF-1. These mutations were located between the transcriptional and translational initiation sites, a region previously shown to be essential for the burst of expression during the very late phase. Addition of partially purified, epitope-tagged VLF-1 to DNA encompassing this "burst sequence" resulted in a shift in the gel electrophoretic mobility of the DNA, indicating that VLF-1 forms a complex with DNA. Addition of an antibody specific for the epitope tag of VLF-1 decreased the mobility of the DNA further, confirming the presence of VLF-1 in the complex. DNase I footprint assays revealed that VLF-1 partially purified from either insect cells or bacterial cells interacted with the burst sequences of both the polh and p10 very-late promoters. Linker scan mutations within the burst sequences severely impaired interaction between VLF-1 and the promoters. We propose that VLF-1 transactivates the polh and p10 promoters by interacting with the burst sequences.

Animals↗
PubMedDate not supplied

Molecular scanning for mutations in the insulin receptor substrate-1 (IRS-1) gene in Mexican Americans with Type 2 diabetes mellitus.

BACKGROUND: Insulin receptor substrate-1 (IRS-1) is an endogenous substrate for the insulin receptor tyrosine kinase, which plays an important role in insulin signaling. Mutations in the IRS-1 gene are associated in some populations with obesity and Type 2 diabetes. METHODS: To determine whether variation in the IRS-1 gene contributes to genetic susceptibility to insulin resistance and Type 2 diabetes in Mexican Americans, the entire coding region of the IRS-1 gene was screened for variation in 31 unrelated subjects with Type 2 diabetes using single-stranded conformational polymorphism analysis (SSCP) and dideoxy sequence analysis. Variants encoding amino acid substitutions were genotyped in 27 unrelated nondiabetic Mexican Americans and in all family members of subjects containing these variants, and association analyses were performed. To trace the ancestral origins of the variants, Iberian Caucasians and Pima Indians were also genotyped. RESULTS: Eight single base changes were found: four silent polymorphisms and four missense mutations (Ala94Thr, Ala512Pro, Ser892Gly and Gly971Arg). Allele frequencies were 0.009, 0.017, 0.017 and 0.043, respectively. There were no significant associations of any of these variants with diabetes, glucose or insulin levels during an oral glucose tolerance test, or with body mass index (BMI) in Mexican American families except for a modest association between the Ala94Thr variant and decreased BMI (30.4 kg/m(2) vs 24.0 kg/m(2); p=0.035). None of these four missense mutations were detected in Pima Indians. In Iberian Caucasians, neither Ala94Thr nor Ser892Gly were detected, and Ala512Pro was detected in only 0/60 diabetic patients and 1/60 nondiabetic controls. Gly971Arg was relatively more common in Iberian Caucasians with 12/58 diabetic patients and 7/60 nondiabetic controls being heterozygous for this variant (p=0.21 for comparison between diabetic and nondiabetic subjects). CONCLUSIONS: Ala94Thr, Ala512Pro and Ser892Gly mutation are rare in the populations studied. Gly971Arg, is more common in Mexican Americans and Caucasians, but is not a major contributor to genetic susceptibility to Type 2 diabetes.

Adult↗
PubMed1995

Scanning for mutations in the human prion protein open reading frame by temporal temperature gradient gel electrophoresis.

Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (CJD) are caused by point mutations or octarepeat insertions in the prion protein (PrP) gene. In the present work a method was established that is appropriate for a thorough screening for mutations in the PrP gene and is generally applicable to screenings of any given gene. Temperature gradient gel electrophoresis (TGGE) was modified at two critical steps by UV cross-linking of the DNA strands and by replacing the spatial gradient with a temporal one. The shift of a DNA band in temporal temperature gradient gel electrophoresis (tTGGE) due to a mutation can be calculated as a function of the position of the mutation in the sequence. Appropriate DNA fragments were selected for polymerase chain reaction (PCR) amplification and analysis by tTGGE on the basis that the predicted band shifts amount to more than 10% of the migration distance for all possible mutations. The accuracy of the prediction was tested experimentally with ten known mutations in the human PrP gene, and quantitative agreement between theory and experiment was achieved. Thus, this screening method is also a suitable means to verify the absence of mutations in a given gene segment.

Base Sequence↗
PubMed1995

Exon scanning for mutations of the NF2 gene in pediatric ependymomas, rhabdoid tumors and meningiomas.

Deletions of chromosome 22 have been identified in 3 types of childhood primary brain tumor: meningiomas, rhabdoid or atypical teratoid tumors (ATT) and ependymomas. This implicates the involvement of tumor suppressor genes on chromosome 22 in the genesis of these rare tumors. One such candidate tumor suppressor gene is the recently cloned neurofibromatosis 2 (NF2) locus. The purpose of our study was to determine the frequency of germ-line and somatic NF2 mutations in a selected group of brain tumors in children. Using single-strand conformation polymorphism (SSCP) assays we screened 17 exons of the NF2 gene in 13 pediatric brain tumors and 9 matched normal blood DNA samples. Tumors included 3 meningiomas, 2 rhabdoid or ATTs, 7 ependymomas and 1 malignant tumor of glial lineage. In addition, lymphoblastoid cell lines from 3 patients with rhabdoid/ATT in whom no tumor tissue was available were analyzed for germ-line mutations. Migration shifts were not detected in any of the normal DNA samples analyzed. Of the 13 tumors screened by SSCP, 1 meningioma with monosomy 22 produced a migration shift in exon 13. DNA sequencing of exon 13 revealed a deletion of a single guanine nucleotide (base 1397) in codon 466, causing a frame shift. While not all mutations might have been picked up by this technique, the data suggest that, similar to adult sporadic meningiomas, some pediatric meningiomas may result from somatic mutations in the NF2 gene. For rhabdoid tumors and ependymomas it appears that a locus distinct from NF2 might be responsible for tumorigenesis.

Base Sequence↗
PubMed1998

Organization of the human carboxypeptidase E gene and molecular scanning for mutations in Japanese subjects with NIDDM or obesity.

Insulin is synthesized in the pancreatic beta cell as a larger precursor molecule proinsulin which is converted to insulin and C-peptide by the concerted action of prohormone convertase 2 (PC2), prohormone convertase 3 (PC3) and carboxypeptidase E (CPE). One of the features of non-insulin-dependent diabetes mellitus (NIDDM) is an elevation in the proinsulin level and/or proinsulin/insulin molar ratio suggesting that mutations in these three proinsulin processing enzymes might contribute to the development of NIDDM. The identification of a mutation in the CPE gene of the fat/fat mouse which leads to marked hyperproinsulinaemia and late-onset obesity and diabetes is consistent with a possible role for mutations in CPE in the development of diabetes and obesity in humans. In order to test this hypothesis, we have isolated and characterized the human CPE gene and screened it for mutations in a group of Japanese subjects with NIDDM and obesity. The human CPE gene consists of 9 exons spanning more than 60 kb. Primer extension analysis identified the transcriptional start site at -141 bp from the translational start site. Single strand conformational polymorphism analysis and nucleotide sequencing of the promoter and entire coding region of the CPE gene in 269 Japanese subjects with NIDDM, 28 nondiabetic obese subjects and 104 nonobese and nondiabetic controls revealed three nucleotide changes, a G-to-T substitution at nucleotide -53, a G-to-A substitution at nucleotide -144 (relative to start of transcription) in the promoter region and a silent G-to-A substitution in codon 219. None of the nucleotide substitutions were associated with NIDDM or obesity. Thus, genetic variation in the CPE gene does not appear to play a major role in the pathogenesis of NIDDM or obesity in Japanese subjects.

Base Sequence↗
PubMed1998

High throughput fluorescence-based conformation-sensitive gel electrophoresis (F-CSGE) identifies six unique BRCA2 mutations and an overall low incidence of BRCA2 mutations in high-risk BRCA1-negative breast cancer families.

Mutational analysis of cancer susceptibility genes has opened up a new era in clinical genetics. In this report we present the results of mutational analysis of the BRCA2 coding sequences in 105 high-risk individuals affected with breast cancer and/or ovarian cancer and previously found to be negative for mutations of the BRCA1 coding sequence in our laboratory. These individuals have a positive family history with three or more cases of breast cancer and/or ovarian cancer at any age from the same side of the family tree. In order to perform a high throughput and reliable mutational analysis of the BRCA genes, we have adapted the conformation-sensitive gel electrophoresis mutation-scanning assay to a fluorescent platform. The advantages are speed, reproducibility and enhanced resolving power of the scanning method. Four unique mutations, including one missense and three frameshift mutations, were identified in the pool of 60 non-Jewish patients (7%). Two cases of the 6174delT mutation were identified in the 45 Ashkenazi Jewish individuals studied (5%). In addition, two novel frameshift mutations, not characteristic of the Jewish subgroup, were identified. Thus there were four mutations in total in this ethnic subgroup (9%). The six mutations identified in this combined patient pool, excluding the 6174delT mutations, are novel and have not been previously reported in the Breast Cancer Information Core (BIC) database. The results indicate that BRCA2 mutations account for the disease in less than 10% of this patient population. In addition, there is no significant difference in frequency of BRCA2 mutations between the Ashkenazi Jewish and non-Jewish families in our clinical patient pool.

Adult↗
PubMed2001

Homogeneous assay based on 52 primer sets to scan for mutations of the ABCA1 gene and its application in genetic analysis of a new patient with familial high-density lipoprotein deficiency syndrome.

Familial high-density lipoprotein (HDL)-deficiency syndromes are caused by mutations of the ABCA1 gene, coding for the ATP-binding cassette transporter 1. We have developed a homogeneous assay based on 52 primer sets to amplify all 50 ABCA1 exons and approximately 1 kb of its promoter. The assay allows for convenient amplification of the gene from genomic DNA and easy mutational analysis through automatic sequencing. It obviates the need to use mRNA preparations, which were difficult to handle and posed a risk to miss splice junction or promoter mutations. The application of the test to the molecular analysis of a new patient with familial HDL-deficiency (Tangier disease) led to a discovery of two novel ABCA1 mutations: C2665del and C4457T.

ATP Binding Cassette Transporter 1↗
PubMed1999

Molecular scanning for mutations in the melanocortin-4 receptor gene in obese/diabetic Japanese.

Decreased function of the melanocortin-4 receptor (MC4R) was reported to cause late-onset obesity and insulin resistance in rodents. Thus mutations in the MC4R gene drew strong attention as a possible cause of obesity and diabetes. We screened for mutations in the MC4R gene in extremely obese [body mass index (BMI) > or = 35 kg/m2] Japanese with diabetes by direct sequencing. A heterozygous mutation (V103I) was detected in one case (2.0 %), however the frequency was not significantly different from that in non-obese (BMI < or = 24 kg/m2) and non-diabetic subjects (2.7 %). No other mutations were detected. These results suggest that mutations including V103I in the MC4R gene are not a major cause of obesity or diabetes in Japanese.

Animals↗