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PubMed2003

Detection of single nucleotide substitution by competitive allele-specific short oligonucleotide hybridization (CASSOH) with immunochromatographic strip.

Recent advances in human genome research have revealed that genetic polymorphisms, such as single nucleotide polymorphisms (SNPs), are closely associated with susceptibility to various common diseases and adverse drug reactions. Also, numerous mutations responsible for a number of genetic diseases have been identified. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We have devised a novel low-tech method for the detection of a single nucleotide substitution using competitive allele-specific short oligonucleotide hybridization with immunochromatographic strip. The gene of interest is PCR-amplified, hybridized to an allele-specific short oligonucleotide probe in the presence of a competitive oligonucleotide, and subjected to chromatography using a DNA test strip at room temperature. The genotype is unambiguously determined by the presence or the absence of visible purple lines on a strip. Feasibility of the method was demonstrated by the detection of a prevalent disease-causing mutations in glycogen storage disease type Ia (G6PC), medium-chain acyl-CoA dehydrogenase deficiency (ACADM), non-ketotic hyperglycinemia (GLDC), and clinically important polymorphisms in the CYP2C19 gene and the aldehyde dehydrogenase 2 gene (ALDH2). The procedure does not demand either technical expertise or expensive instruments and is readily performed in local clinical laboratories. The result is obtained within 10 min after PCR. This rapid and simple method of SNP detection may be used for point-of-care genetic diagnosis with potentially diverse clinical applications. Hum Mutat 22:166-172, 2003.

Alleles↗
PubMed2003

High fidelity SNP genotyping using sequence-specific primer elongation and fluorescence correlation spectroscopy.

Reliable, efficient and cost-effective modalities are urgently needed for mass screening of gene mutations. Previous reports have shown that SSCP or genechip methods require substantial time and monetary costs, thus limiting their appeal. Sequence Specific Primer Polymerase Chain Reaction (SSP-PCR) is a reliable and cost-effective method that utilizes the 3'-end discrimination properties of polymerase. However, the applicability of conventional SSP-PCR is limited due to the difficulties associated with determining optimal conditions and because mis-matched primers are amplified, resulting in signal noise during end-point assay. To overcome this problem, we eliminated the reverse primers from SSP-PCR, thus preventing amplification of mis-matched primers. We designated this method Sequence-Specific Primer Cycle Elongation (SSPCE). However, the detection of elongated sequence specific primers was difficult using conventional electrophoresis due to the small amounts of amplification product present. We therefore combined SSPCE and Fluorescence Correlation Spectroscopy, which is a novel technique used to determine the number and size of fluorophores at nano-molar concentrations, and designated the method SSPCE-FCS. We compared conventional SSP-PCR and SSPCE-FCS with regard to determining optimal conditions using two Mitochondrial SNPs (G --> A at position 1598, G --> A at position 12192). We were able to determine the optimal conditions for the SNP at position 1598 using either method. However, optimal conditions could only be determined for SSPCE-FCS with the 12192 mutation because non-specific amplification was observed at a wide range of annealing temperatures in SSP-PCR. We then applied this method to three other SNPs and the results were consistent with the results of sequencing data.

Base Sequence↗
PubMed2006

Mutation analysis of hCDC4 in AML cells identifies a new intronic polymorphism.

hCDC4 (FBW7, FBXW7) is a new potential tumor suppressor gene which provides substrate specificity for SCF (Skp-Cullin-F-box) ubiquitin ligases and thereby regulates the degradation of potent oncogenes such as cyclin E, Myc, c-Jun and Notch. Mutations in the hCDC4 gene have been found in several solid tumors such as pancreas, colorectal or endometrial cancer. We carried out a mutation analysis of the hCDC4 gene in 35 samples of patients with Acute Myeloid Leukemia (AML) to elucidate a possible role of hCDC4 mutations in this disease. By direct DNA sequencing and digestion with Surveyor nuclease one heterozygous mutation in the 5' untranslated region of exon 1, transcript variant 3 was detected. Additionally, we could identify a new intronic SNP downstream of exon 10. The new variation was present in 20% of AML samples and was furthermore confirmed in a panel of 51 healthy individuals where it displayed a frequency of 14%. In conclusion we provide first data that in contrast to several solid tumors, mutations in the hCDC4 gene may not play a pivotal role in the pathogenesis of AML. Furthermore, we describe a new intronic polymorphism with high frequency in the intron sequence of the hCDC4 gene.

Journal Article↗
PubMed2006

Genetic variants of the NOTCH3 gene in migraine--a mutation analysis and association study.

Mutations in the NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Exons 3 and 4 are mutation hotspots. Migraine is a clinical hallmark of CADASIL. The objective of this study was to investigate whether genetic variants in exons 3 and 4 of the NOTCH3 gene are associated with migraine. Exons 3 and 4 of the NOTCH3 were analysed for mutations and polymorphisms by direct DNA sequencing in 97 migraineurs and the same number of control individuals. No mutations in exons 3 and 4 of the NOTCH3 gene were found in 97 patients with migraine. However, association analysis revealed significant association of the single nucleotide polymorphism (SNP) rs1043994 with migraine.

Adolescent↗
PubMed2002

A novel mutation in the IHH gene causes brachydactyly type A1: a 95-year-old mystery resolved.

Brachydactyly type A1 (BDA1) was the first disorder described in terms of autosomal dominant Mendelian inheritance. Early in the 1900s Farabee and Drinkwater described a number of families with BDA1. Examination of two of Drinkwater's families has revealed that, although they are not known to be related, both share a common mutation within the Indian hedgehog gene ( IHH). This novel mutation is a guanine to adenine transition at nucleotide 298, resulting in an Asn100Asp amino acid substitution. Both families demonstrate significant intrafamilial phenotypic heterogeneity among the affected individuals. Examination of single nucleotide polymorphisms (SNP) has shown that the affected individuals in both families share SNPs within IHH consistent with that of a common founder. The identification of the same mutation in these families has answered a question that is nearly a century old about the genetic cause of their disease and supports the hypothesis that IHH plays a pivotal role in normal human skeletogenesis.

Amino Acid Sequence↗
PubMed2000

A common polymorphism associated with antibiotic-induced cardiac arrhythmia.

Drug-induced long QT syndrome (LQTS) is a prevalent disorder of uncertain etiology that predisposes to sudden death. KCNE2 encodes MinK-related peptide 1 (MiRP1), a subunit of the cardiac potassium channel I(Kr) that has been associated previously with inherited LQTS. Here, we examine KCNE2 in 98 patients with drug-induced LQTS, identifying three individuals with sporadic mutations and a patient with sulfamethoxazole-associated LQTS who carried a single-nucleotide polymorphism (SNP) found in approximately 1.6% of the general population. While mutant channels showed diminished potassium flux at baseline and wild-type drug sensitivity, channels with the SNP were normal at baseline but inhibited by sulfamethoxazole at therapeutic levels that did not affect wild-type channels. We conclude that allelic variants of MiRP1 contribute to a significant fraction of cases of drug-induced LQTS through multiple mechanisms and that common sequence variations that increase the risk of life-threatening drug reactions can be clinically silent before drug exposure.

Amino Acid Sequence↗
PubMed2001

Novel SNP at the common primer site of exon IIIa of FGFR2 gene causes error in molecular diagnosis of craniosynostosis syndrome.

Most mutations in Crouzon, Pfeiffer, and Apert syndromes are in the extracellular, third immunoglobulin-like domain and adjacent linker regions (exons IIIa and IIIc) of the fibroblast growth factor receptor 2 (FGFR2) gene. Using the published primers for PCR, a patient with Crouzon syndrome was found to be homozygous for a mutation that results in a Q289P amino acid substitution in FGFR2. Two additional patients; one with Apert syndrome and P253R mutation, the other with Pfeiffer syndrome and S267P mutation, also appeared to be homozygous. Using a new primer located 146 bp 5' of exon IIIa for PCR followed by sequencing revealed an A to G polymorphism at -62 [corrected] position of exon IIIa. All three patients were heterozygous for both the mutation and the polymorphism. These results indicate that the polymorphism and the mutation are not on the same chromosome. The single nucleotide polymorphism is located at the second to the last base of the 3' end of the published primer. This primer mismatch caused the failure of amplification of the normal chromosome and thus, the apparent homozygosity. The frequency of this novel polymorphism was determined to be 0.03 by studying 326 chromosomes from the general population. We propose that a new primer should be used for mutational analysis of exon IIIa of FGFR2 to avoid misdiagnosis caused by primer mismatch.

Base Sequence↗
PubMed2006

[Effects of MTNR1A gene on litter size in a large white and a landrace herd].

Two pairs of primers were designed based on the known sequence in GenBank for amplification of MTNR1A gene in a Large White and a Landrace herd. Using PCR-SSCP (single strand conformation polymorphism), we found a single nucleotide polymorphism(SNP) within the product amplified from the first pair of primers. PCR products from randomly selected different genotypes were sequenced after were recovered and purified. Results revealed a synonymous single base mutation(G-->A) at +159bp(sequence numbering based on Genbank accession number U73326) for the BB genotype. When analyzed for association with litter size traits, this MTNR1A SNP was found to have no significant effect on litter size traits.

Animals↗
PubMed2006

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy.

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5' end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled ('cold') oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10(-8) M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10(-11) M solutions of PCR amplicons from M.tuberculosis in only 100 s.

Bacterial Proteins↗
PubMed2005

Decreased E-cadherin expression correlates with higher stage of Wilms' tumors.

PURPOSE: The aim of this study was to examine the association between E-cadherin expression and markers of Wilms' tumor aggression, including metastasis and recurrence. METHODS: Forty Wilms' tumor samples from the National Wilms' Tumor Study Group underwent immunohistochemical staining for E-cadherin. Tumor stage at diagnosis, recurrence, and loss of heterozygosity at 16q status was known for each of the tumor samples. E-Cadherin cell staining was defined as high (>33%) or low (<33%), and values were assigned by a pathologist blinded to the tumor characteristics. Five stage IV tumors were ineligible for assay because of lack of a tubular component. To identify a mechanism of downregulation, we screened tumor DNA for genetic mutations in exons 1-16 using a combination of WAVE and sequence analysis. To assess the functional significance of the identified mutations, the authors compared amino acid homology across multiple species. Finally, they performed reverse transcriptase-polymerase chain reaction for those tumors with intronic single nucleotide polymorphisms (SNPs) to evaluate for mRNA splice variants. RESULTS: Wilms' tumors presenting with metastatic (stage IV) disease demonstrated decreased levels of E-cadherin expression compared with localized tumors (stage I) (Fisher's Exact test, P < .01). In a search for the mechanism of the downregulation of E-cadherin, we identified 5 different mutations in 7 high stage tumors (7/15) and 1 mutation in a low stage tumor (1/20). The mutations occurred in amino acids that were conserved across multiple species. Additionally, 11 of 15 high stage tumors contained an intronic SNP located within 6 bp of the 5 intronic splice junction immediately downstream of exon 1. However, examination of 5 of these tumors using reverse transcriptase-polymerase chain reaction showed that this intronic SNP does not appear to disrupt the assembly of full-length E-cadherin transcripts. Lastly, no correlation was identified between E-cadherin expression and recurrence of disease. CONCLUSIONS: In this study, the authors have found an association between decreased E-cadherin expression and metastatic Wilms' tumor. Mutations identified may help identify a mechanism for downregulation. The functional significance of these mutations is supported by the conserved nature of the amino acids across multiple species. The authors believe these findings support the involvement of E-cadherin in the evolution of Wilms' tumor.

Cadherins↗
PubMed2002

Determination of the factor V Leiden single-nucleotide polymorphism in a commercial clinical laboratory by use of NanoChip microelectronic array technology.

BACKGROUND: Methods for analysis of the single-nucleotide polymorphism (SNP) known as factor V Leiden (FVL) are described. The technique provides rapid, highly accurate detection of the point mutation that encodes for replacement of arginine-506 with glutamine. After formal assay qualification, 758 clinical samples that had previously been analyzed by the Invader Monoplex Assay were tested as research samples in a commercial clinical laboratory. METHODS: Primers specific for factor V (FV) were prepared, and PCR was performed. Samples were analyzed using the NanoChip Molecular Biology Workstation with fluorescently labeled reporters for wild-type and SNP sequences. RESULTS: Of the 635 samples classified by the Third Wave assay as FV wild type, 10 were identified as heterozygous FVL by the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were wild type, and of the 9 reported homozygous samples, 6 were homozygous, 2 were heterozygous, and 1 was FV wild type by the NanoChip assay. All 17 results that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctly classified by the NanoChip technology. The Nanochip system was 100% accurate in characterizing wild-type, heterozygous, and homozygous samples compared with accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analysis. CONCLUSIONS: The NanoChip microelectronic chip array technology is an accurate and convenient method for FVL screening of research samples in a clinical laboratory environment.

Autoanalysis↗
PubMed2003

Sequence diversity of KIAA0027/MLC1: are megalencephalic leukoencephalopathy and schizophrenia allelic disorders?

The aim of the study is to validate the etiological role of KIAA0027/MLC1 in childhood-onset megalencephalic leukoencephalopathy with subcortical cysts (MLC) and in schizophrenia, particularly the catatonic subtype, which were reported to be allelic diseases. Among a series of five patients with MLC, four mutant alleles were detected: one case of compound heterozygosity for a splice site mutation and a six-base-pair in-frame deletion, one patient with a homozygous frameshifting insertion-deletion, and a further case heterozygous for a A157E substitution. A systematic mutation screening in 140 index cases with schizophrenia revealed 13 different single nucleotide polymorphisms (SNPs): one SNP in the 5'-UTR, seven SNPs in intronic regions, two synonymous codon variants (T52, Y199), and three coding variants. Two of them, C171F and N218K, were observed in controls at a significant frequency. The L309M variant that was previously supposed to be the causative factor for chromosome 22q(tel) linked-periodic catatonia was found nonsegregating in a further multiplex pedigree. Furthermore, a complicated 33-bp insertion/deletion polymorphism at the 5'-end of exon 11 of MLC1 was found at equal frequency among schizophrenic patients and controls. In summary, our study provides further evidence for allelic heterogeneity in megalencephalic leukoencephalopathy, excludes MLC1 as a susceptibility locus for schizophrenia, and thereby rules out that MLC and schizophrenia are allelic disorders.

Adolescent↗
PubMed2002

Allelic imbalances in human bladder cancer: genome-wide detection with high-density single-nucleotide polymorphism arrays.

BACKGROUND: Bladder cancer is characterized by genomic instability. In this study, we investigated whether genome-wide screening using single-nucleotide polymorphism (SNP) arrays could detect allelic imbalance (loss or gain of at least one allele) in bladder cancers. METHODS: For microarray analysis, DNA was isolated from microdissected bladder tumors and leukocytes from 11 patients. The stage T1 tumor (connective tissue invasive) and the subsequent stage T2-4 tumor (muscle invasive) were available from eight of these patients, and only the first muscle-invasive stage T2-4 tumor was available from three of the 11 patients. The microarray contained 1494 biallelic polymorphic sequences. For microsatellite analyses, DNA was isolated from tumors and leukocytes of nine patients with primary T2-4 tumors and 13 patients with Ta (noninvasive) tumors. All statistical tests were two-sided. RESULTS: We assigned a genotype to 1204 loci, 343 of which were heterozygous. Allelic imbalance was detected in known areas of imbalance on chromosomes 6, 8, 9, 11, and 17, and a new area of imbalance was detected on the p arm of chromosome 6. Microsatellite analysis of nine other T2-4 tumors and 13 Ta tumors showed that allelic imbalance was more frequent in T2-4 tumors than in Ta tumors (P<.001). We detected 8.5 allelic imbalances (median) in 348 informative loci in T1 tumors and 28 allelic imbalances (median) in 329 informative loci in T2-4 tumors. When pairs of T1 and T2-4 tumors were analyzed from eight patients, 68% of imbalances detected in T1 tumors (146 imbalances) occurred in the subsequent T2-4 tumors (99 imbalances). Homozygous TP53 mutations were more often associated (P =.005) with high allelic imbalance than with low allelic imbalance. CONCLUSION: SNP arrays are feasible for high-throughput, genome-wide scanning for allelic imbalances in bladder cancer.

Allelic Imbalance↗
PubMed2004

Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye.

BACKGROUND: Homogeneous PCR methods for genotyping usually require fluorescently labeled oligonucleotide probes. Amplicon melting with the DNA dye LCGreen I was recently introduced as a closed-tube method of genotyping that does not require probes or real-time PCR. However, some single-nucleotide polymorphisms (SNPs) could not be completely genotyped without addition of a known genotype, and high-resolution melting techniques were necessary. METHODS: A 3'-blocked, unlabeled oligonucleotide probe and the saturating dye, LCGreen I, were added to standard PCR reagents before amplification. After PCR, the samples were melted at 0.1-0.3 degrees C/s in high-resolution (HR-1), high-throughput (LightTyper), and rapid-cycle, real-time (LightCycler) instruments, and fluorescence melting curves were recorded. RESULTS: Derivative melting curves of the probe-target duplexes were characteristic of the genotype under the probe. With synthetic plasmid templates, all SNP base combinations could be genotyped. For human genomic DNA, the technique was demonstrated with mutations associated with cystic fibrosis, including SNPs (G542X, I506V, and F508C) and 3-bp deletions (F508del and I507del). CONCLUSIONS: Genotyping of SNPs and small deletions by melting analysis of an unlabeled probe in the presence of LCGreen I is simple and rapid. Only three unlabeled oligonucleotides (two primers and one probe), a saturating DNA dye, PCR, and a melting instrument are required. The method is closed-tube, does not require fluorescently labeled probes or real-time PCR, and can be completed in <10 min on any instrument capable of monitoring melting curves by fluorescence.

Coloring Agents↗
PubMed2003

Impact of purified water quality on molecular biology experiments.

Purified water is a reagent used in a variety of molecular biology experiments, for sample and media preparation, in mobile phases of liquid chromatography techniques, and in rinsing steps. The combination of several technologies in water purification systems allows delivering high-purity water adapted to each application and technique. Through a series of examples, the importance of water quality on biotechnology experiments, such as single nucleotide polymorphism (SNP) analysis by denaturating HPLC, RNA preparation and PCR, is presented. Results obtained on DNA mutation and single nucleotide polymorphism analysis using the denaturating HPLC (DHPLC) technique highlight the benefits of organic removal by UV photooxidation process. Comparative gel electrophoresis data show that ultrafiltration is as efficient as diethylpyrocarbonate (DEPC) treatment for suppressing RNase activity in water. Gel electrophoresis and densitometry measurement also point out the benefits of ultrafiltration to carry out reverse transcriptase-polymerase chain reaction quantitatively.

Animals↗
PubMed2006

An effect of a missense mutation in the porcine melanocortin-4 receptor (MC4R) gene on production traits in Polish pig breeds is doubtful.

A missense G-A SNP in the porcine melanocortin-4 receptor (MC4R) gene that causes an Asp-Asn substitution at position 298 of the corresponding MC4R protein is considered to be economically important, although published results on its effect are inconsistent. We analysed the association of this MC4R polymorphism with production traits in 679 gilts from two breeds, Polish Large White (PLW) and Polish Landrace (PL), as well as one synthetic line 990. The frequency of the A allele differed significantly among the breeds with frequencies of 0.76, 0.29 and 0.16 in PLW, PL and line 990 respectively. There was no evidence of an effect of this polymorphism on daily food intake, backfat thickness or abdominal fat. The A allele was correlated with higher test daily gains and lower levels of intramuscular fat in PL, and increased levels of intramuscular fat in PLW.

Animals↗
PubMed2003

SNP and haplotype variation in the human genome.

We have surveyed and summarized several aspects of DNA variability among humans. The variation described is the result of mutation followed by a combination of drift, migration and selection bringing the frequencies high enough to be observed. This paper describes what we have learned about how DNA variability differs among genes and populations. We sequenced functional regions of a set of 3950 genes. DNA was sampled from 82 unrelated humans: 20 African-Americans, 20 East Asians, 21 Caucasians, 18 Hispanic-Latinos and 3 Native Americans. Different aspects of variability showed a great deal of concordance. In particular, we studied patterns of single nucleotide polymorphism (SNP) allele and haplotype sharing among the four, large sample populations. We also examined how linkage disequilibrium (LD) between SNPs relates to physical distance in the different populations. It is clear from our findings that while many variants are common to all populations, many others have a more restricted distribution. Research that attempts to find genetic variants that explain phenotypic variants must be careful in their choice of study population.

Alleles↗
PubMed2006

Fine mapping of the keratoconus with cataract locus on chromosome 15q and candidate gene analysis.

PURPOSE: To report the fine mapping of the keratoconus with cataract locus on chromosome 15q and the mutational analysis of positional candidate genes. METHODS: Genotyping of two novel microsatellite markers and a single nucleotide polymorphism (SNP) in the critical region of linkage for keratoconus with cataract on 15q was performed. Positional candidate genes (MORF4L1, KIAA1055, ETFA, AWP1, REC14, KIAA1199, RCN2, FAH, IDH3A, MTHFS, ADAMTS7, MAN2C1, PTPN9, KIAA1024, ARNT2, BCL2A1, ISL2, C15ORF22 (P24B), DNAJA4, FLJ14594, CIB2 (KIP2), C15ORF5, and PSMA4) prioritized on the basis of ocular expression and probable function were screened by PCR-based DNA sequencing methods. RESULTS: We report the refinement of the linkage region for keratoconus with cataract to an interval of approximately 5.5 Mb flanked by the MAN2C1 gene and the D15S211 marker on chromosome 15q. Mutational analysis of positional candidate genes detected many sequence variations and single nucleotide polymorphisms. None of the sequence variants were considered pathogenic as they were also found in unaffected family members and normal control DNA samples. CONCLUSIONS: Fine mapping of the keratoconus with cataract locus on 15q has reduced the linked region to 5.5 Mb, thereby excluding 28 candidate genes. A further 23 candidate genes were excluded by direct sequencing methods, although a pathogenic genomic rearrangement or exonic deletion would not have been detected.

Case-Control Studies↗