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PubMed2005

Association of Plasmodium falciparum isolates encoding the p. Falciparum chloroquine resistance transporter gene K76T polymorphism with anemia and splenomegaly, but not with multiple infections.

The aim of the study was to assess whether infections with Plasmodium falciparum isolates encoding the P. falciparum chloroquine resistance transporter (pfcrt) gene K76T polymorphism, a molecular marker for chloroquine resistance, are associated with multiple infections, age, or clinical signs of malaria in a semi-immune population in a holoendemic area of Burkina Faso. The parameters of interest were investigated in 210 P. falciparum-positive inhabitants. Logistic regression analysis showed that pfcrt K76T-carrying isolates are significantly more likely to cause anemia and splenomegaly. Furthermore, we found that infections with P. falciparum isolates encoding pfcrt K76T are dependent on age rather than multiple infections. Our findings suggest that pfcrt K76T might serve as a valuable marker for assessing the long-term clinical effect of chronic infections with chloroquine-resistant P. falciparum isolates in populations, without the need of drug efficacy trials.

Adult↗
PubMed2005

MutDB services: interactive structural analysis of mutation data.

Non-synonymous single nucleotide polymorphisms (SNPs) and mutations have been associated with human phenotypes and disease. As more and more SNPs are mapped to phenotypes, understanding how these variations affect the function and expression of genes and gene products becomes an important endeavor. We have developed a set of tools to aid in the understanding of how amino acid substitutions affect protein structures. To do this, we have annotated SNPs in dbSNP and amino acid substitutions in Swiss-Prot with protein structural information, if available. We then developed a novel web interface to this data that allows for visualization of the location of these substitutions. We have also developed a web service interface to the dataset and developed interactive plugins for UCSF's Chimera structural modeling tool and PyMOL that integrate our annotations with these sophisticated structural visualization and modeling tools. The web services portal and plugins can be downloaded from http://www.lifescienceweb.org/ and the web interface is at http://www.mutdb.org/.

Amino Acid Substitution↗
PubMed2005

Polymorphism and double hexamer structure in the archaeal minichromosome maintenance (MCM) helicase from Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM), a cellular replicative helicase, is a useful model for the more complex eukaryotic MCMs. Biochemical and crystallographic evidence indicates that mtMCM assembles as a double hexamer (dHex), but previous electron microscopy studies reported only the presence of single heptamers or single hexamers (Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M., and Onesti, S. (2003) EMBO Rep. 4, 1079-1083; Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K., and Egelman, E. H. (2002) EMBO Rep. 3, 792-797). Here we present the first three-dimensional electron microscopy reconstruction of the full-length mtMCM dHex in which two hexamers contact each other via the structurally well defined N-terminal domains. The dHex has obvious side openings that resemble the side channels of LTag (large T antigen). 6-fold and 7-fold rings were observed in the same mtMCM preparation, but we determined that assembly as a double ring favors 6-fold structures. Additionally, open rings were also detected, which suggests a direct mtMCM loading mechanism onto DNA.

Chromosomes, Archaeal↗
PubMed1995

Restriction fragment length polymorphism of rRNA genes for molecular typing of members of the family Legionellaceae.

Typing of Legionella pneumophila remains important in the investigation of outbreaks of Legionnaires' disease and in the control of organisms contaminating hospital water. We found that the discriminatory power of a nonradioactive ribotyping method could be improved by combining results obtained with four restriction enzymes (HindIII, NciI, ClaI, and PstI). Fifty-eight clinical and environmental L. pneumophila strains including geographically unrelated as well as epidemiologically connected isolates were investigated. Epidemiologically related strains had the same ribotypes independent of the combinations of enzymes used. Some strains belonging to the same serogroup were assigned to different ribotypes, and some ribotypes contained members of different serogroups, indicating, as others have found, that serogroup and genotype are not always related. The discriminatory power of the method was estimated by calculating an index of discrimination (ID) for individual enzymes and combinations thereof. The combined result with all four enzymes was highly discriminatory (ID = 0.97), but results for three enzymes also yielded ID values acceptable for epidemiological purposes. In addition, the testing of 27 type strains and 6 clinical isolates representing Legionella species other than L. pneumophila indicated that ribotyping might be of value for species identification within this genus, as previously suggested.

Bacterial Typing Techniques↗
PubMed2002

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters.

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.

Bacterial Typing Techniques↗
PubMed1995

Comparative analysis of HLA polymorphism at the serologic and molecular level in Moroccan and Ashkenazi Jews.

The Jewish people comprise two major groups, one encompassing the Jews of Ashkenazi (Central and Eastern European) origin and the other including those of Sephardic (Middle Eastern and North African) descent. To the latter belong the Jews of Moroccan stock, who form the largest Jewish subgroup among the non-Ashkenazi population living in Israel. As the members of each of these groups differ in physiognomy and life style, it was of interest to investigate whether these differences are also reflected in their respective HLA compositions. To this end, 132 subjects of Ashkenazi and 113 individuals of Moroccan origin residing in Israel were tested and the results compared with data for other populations made available by the 11th International Histocompatibility Workshop. Comparison between their HLA profiles and those of non-Jews revealed that the Jewish groups in some aspects resembled one another but in others showed disparities. The dissimilarities between the various groups are expressed in terms of gene and haplotype frequencies, as well as in HLA-disease associations (as for example rheumatoid arthritis, erosive lichen planus, primary Sjögren's syndrome, pemphigus vulgaris). However, both Jewish groups shared some unique features with respect to HLA class II allelic frequencies, pointing to a common ancestry.

Adolescent↗
PubMed2001

Prediction of deleterious human alleles.

Single nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation, occurring with an average density of approximately 1/1000 nucleotides of a genotype. SNPs are either neutral allelic variants or are under selection of various strengths, and the impact of SNPs on fitness remains unknown. Identification of SNPs affecting human phenotype, especially leading to risks of complex disorders, is one of the key problems of medical genetics. SNPs in protein-coding regions that cause amino acid variants (non-synonymous cSNPs) are most likely to affect phenotypes. We have developed a straightforward and reliable method based on physical and comparative considerations that estimates the impact of an amino acid replacement on the three-dimensional structure and function of the protein. We estimate that approximately 20% of common human non-synonymous SNPs damage the protein. The average minor allele frequency of such SNPs in our data set was two times lower than that of benign non-synonymous SNPs. The average human genotype carries approximately 10(3) damaging non-synonymous SNPs that together cause a substantial reduction in fitness.

Alleles↗
PubMed2004

Pyrene-labeled base-discriminating fluorescent DNA probes for homogeneous SNP typing.

This paper describes the design of novel base-discriminating fluorescent (BDF) nucleobases and their application to single nucleotide polymorphism (SNP) typing. We devised novel BDF nucleosides, (Py)U and (Py)C, which contain a pyrenecarboxamide chromophore connected by a propargyl linker. The fluorescence spectrum of the duplex containing a (Py)U/A base pair showed a strong emission at 397 nm on 327 nm excitation. In contrast, the fluorescence of duplexes containing (Py)U/N base pairs (N = C, G, or T) was considerably weaker. The proposed structure of the duplex containing a matched (Py)U/A base pair suggests that the high polarity near the pyrenecarboxamide group is responsible for the strong A-selective fluorescence emission. Moreover, the fluorescence of the duplex containing a (Py)U/A base pair was not quenched by a flanking C/G base pair. The fluorescence properties are quite different from previous BDF nucleobases, where fluorescence is quenchable by flanking C/G base pairs. The duplex containing the C derivative, (Py)C, selectively emitted fluorescence when the base opposite (Py)C was G. The drastic change of fluorescence intensity by the nature of the complementary base is extremely useful for SNP typing. (Py)U- and (Py)C-containing oligodeoxynucleotides acted as effective reporter probes for homogeneous SNP typing of DNA samples containing c-Ha-ras and BRCA2 SNP sites.

Amides↗
PubMed2004

topoSNP: a topographic database of non-synonymous single nucleotide polymorphisms with and without known disease association.

The database of topographic mapping of Single Nucleotide Polymorphism (topoSNP) provides an online resource for analyzing non-synonymous SNPs (nsSNPs) that can be mapped onto known 3D structures of proteins. These include disease- associated nsSNPs derived from the Online Mendelian Inheritance in Man (OMIM) database and other nsSNPs derived from dbSNP, a resource at the National Center for Biotechnology Information that catalogs SNPs. TopoSNP further classifies each nsSNP site into three categories based on their geometric location: those located in a surface pocket or an interior void of the protein, those on a convex region or a shallow depressed region, and those that are completely buried in the interior of the protein structure. These unique geometric descriptions provide more detailed mapping of nsSNPs to protein structures. The current release also includes relative entropy of SNPs calculated from multiple sequence alignment as obtained from the Pfam database (a database of protein families and conserved protein motifs) as well as manually adjusted multiple alignments obtained from ClustalW. These structural and conservational data can be useful for studying whether nsSNPs in coding regions are likely to lead to phenotypic changes. TopoSNP includes an interactive structural visualization web interface, as well as downloadable batch data. The database will be updated at regular intervals and can be accessed at: http://gila.bioengr.uic.edu/snp/toposnp.

Computational Biology↗
PubMed2005

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora).

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.

Animals↗
PubMed2001

Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos.

CYP3A4 is the most abundant isoform of cytochrome P450 (CYP) in adult human liver. It metabolizes numerous clinically, physiologically, and toxicologically important compounds. The expression of CYP3A4 varies 40-fold in individual human livers, and metabolism of CYP3A4 substrates varies at least 10-fold in vivo. Single nucleotide polymorphisms (SNPs) in CYP3A4 were identified by direct sequencing of genomic DNA in 72 individuals from three different ethnic groups, including Caucasians, Blacks (African-Americans and African pygmies), and Asians. A total of 28 SNPs were identified, including five which produced coding changes M445T (CYP3A4*3), R162Q (CYP3A4*15), F189S (CYP3A4*17), L293P (CYP3A4*18), and P467S (CYP3A4*19). The latter four represent new alleic variants. Racial variability was observed for the frequency of individual SNPs. CYP3A R162Q was identified only in Black populations with an allelic frequency of 4%. CYP3A4 F189S and CYP3A4 M445T were identified in Caucasians with allelic frequencies 2% and 4%, respectively. L293P and P467S were only observed in Asians at allelic frequencies of 2%. The cDNAs for the F189S, L293P, M445T, and P467S mutant alleles were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Testosterone and the insecticide chlorpyrifos were used to assess the catalytic activities of the most common CYP3A4 allele (CYP3A4*1) and its allelic variants. CYP3A4 F189S exhibited lower turnover numbers for testosterone and chlorpyrifos, while CYP3A4 L293P had higher turnover numbers for both substrates. The turnover numbers of the CYP3A4 M445T and P467S alleles to metabolize these compounds were not significantly different from those of wild-type CYP3A4.

Alleles↗
PubMed2003

Structural location of disease-associated single-nucleotide polymorphisms.

Non-synonymous single-nucleotide polymorphism (nsSNP) of genes introduces amino acid changes to proteins, and plays an important role in providing genetic functional diversity. To understand the structural characteristics of disease-associated SNPs, we have mapped a set of nsSNPs derived from the online mendelian inheritance in man (OMIM) database to the structural surfaces of encoded proteins. These nsSNPs are disease-associated or have distinctive phenotypes. As a control dataset, we mapped a set of nsSNPs derived from SNP database dbSNP to the structural surfaces of those encoded proteins. Using the alpha shape method from computational geometry, we examine the geometric locations of the structural sites of these nsSNPs. We classify each nsSNP site into one of three categories of geometric locations: those in a pocket or a void (type P); those on a convex region or a shallow depressed region (type S); and those that are buried completely in the interior (type I). We find that the majority (88%) of disease-associated nsSNPs are located in voids or pockets, and they are infrequently observed in the interior of proteins (3.2% in the data set). We find that nsSNPs mapped from dbSNP are less likely to be located in pockets or voids (68%). We further introduce a novel application of hidden Markov models (HMM) for analyzing sequence homology of SNPs on various geometric sites. For SNPs on surface pocket or void, we find that there is no strong tendency for them to occur on conserved residues. For SNPs buried in the interior, we find that disease-associated mutations are more likely to be conserved. The approach of classifying nsSNPs with alpha shape and HMM developed in this study can be integrated with additional methods to improve the accuracy of predictions of whether a given nsSNP is likely to be disease-associated.

Disease↗
PubMed2001

Molecular typing methods for the epidemiological identification of Clostridium difficile strains.

Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhea (CDAD), the most common cause of nosocomial diarrhea. Cross-infection between patients and transmission through the environment and medical personnel are important factors in the acquisition of CDAD. In order to understand differences in epidemiology and pathogenesis, a number of typing schemes have been developed. We will review the typing methods used to study the epidemiology of C. difficile infections and how they have evolved from a phenotypic identification to state of the art molecular methods, detecting genetic polymorphisms among strains. These molecular methods include PCR-based methods (arbitrarily primed-PCR [AP-PCR] and PCR ribotyping), restriction endonuclease analysis (REA) and pulse field gel electrophoresis (PFGE). The application, usefulness and feasibility of these methods are compared and discussed. Finally, the role of genomics as a tool to investigate CDAD is introduced.

Bacterial Typing Techniques↗
PubMed2000

Genetic polymorphism of thiopurine S-methyltransferase: molecular mechanisms and clinical importance.

The activity of thiopurine S-methyltransferase (TPMT) is inherited as an autosomal co-dominant trait. In most large world populations studied to date, approximately 10% of the population have intermediate activity due to heterozygosity at the TPMT locus, and about 0.33% is TPMT deficient. TPMT is now one of the most well characterized genetic polymorphisms of drug metabolism, with the genetic basis having been well defined in most populations, providing molecular strategies for studying this genetic polymorphism in human and experimental models. Three mutant alleles, TPMT(*)2, TPMT(*)3A and TPMT(*)3C, account for the great majority of mutant alleles in all human populations studied to date. Significant ethnic differences occur in the frequencies of these mutant alleles. Progress in DNA analysis has made it practical to use genotyping techniques for the molecular diagnosis of TPMT deficiency and heterozygosity, thereby avoiding adverse effects that are more prevalent in TPMT-deficient and heterozygous patients prescribed thiopurine medications.

Animals↗
PubMed1997

Molecular analysis of HLA-DR polymorphism in polymyalgia rheumatica. Swiss Group for Research on HLA in Polymyalgia Rheumatica.

OBJECTIVE: To analyze by molecular typing possible associations of HLA-DRB1 alleles with polymyalgia rheumatica (PMR) compared to controls and patients with rheumatoid arthritis (RA) in Switzerland. METHODS: In a multicenter survey, we recruited 100 patients with PMR with and without signs of giant cell arteritis (GCA), 198 with RA, and 200 controls (volunteer bone marrow donors). HLA-DR generic typing was performed by microtiter plate oligotyping and DR4 subtypes analyzed by dot blot hybridization with sequence specific oligonucleotides or by polymerase chain reaction sequence specific primers. RESULTS: DR4 and DR1 tended to be increased in PMR, compared to controls (36 vs 30%, p = 0.30; and 19 vs 12%, p = 0.16, respectively). Frequencies of all RA associated DR4 and DR1 subtypes tended to be increased in PMR as well. Frequency of the HLA-DR beta 1 70-74 shared motif (QK/RRAA) was significantly higher in PMR than in controls [50 vs 36%, odds ratio (OR) = 1.8, p = 0.018], although lower than in RA (77 vs 36%, OR = 6.0, p < 0.0001), and slightly out of the range of significance if a Bonferroni correction was applied (p = 0.1). At double dose, this epitope was also increased in PMR, but not significantly (5 vs 2%, OR = 2.6, p = 0.17), while it was markedly augmented in RA (22 vs 2%, OR = 14, p = < 0.0001). In patients with the shared epitope, the frequency of clinical signs of GCA tended to be increased (19 vs 10%, p = 0.25). Frequency of the HLA-DR beta 1 DRYF 28-31 motif was identical in PMR (95%) and controls (93%). CONCLUSION: PMR may be associated with the HLA-DR beta 1 70-74 shared epitope. This association, however, would be much weaker for PMR than for RA, particularly with the shared epitope at double dose. PMR is clearly not associated with the HLA-DR beta 1 DRYF 28-31 motif.

Aged↗
PubMed2005

Testing of amplified fragment length polymorphism (AFLP) technique as a tool for molecular epidemiology of Trichinella nativa.

A total of nine Trichinella nativa isolates were compared by amplified fragment length polymorphism (AFLP). Four hundred nanograms of genomic DNA from a pool of 10--20 larvae were digested using HindIII and MseI restriction endonucleases. Of the 16 primer combinations initially tested, Hind-C and Mse-C primers showed rich polymorphism with approximately 40--90 bands in the range of 30--270 bp. Genetic similarities were estimated visually. AFLP provided discriminatory banding patterns and may therefore be used as a method for detecting variation in T. nativa populations. However, the heterogeneous patterns obtained from pooled samples emphasize the need for further development of the sampling and numerical analysis of the patterns for epidemiological and taxonomical interpretation.

Animals↗
PubMed2003

Rapid detection of aneuploidy (trisomy 21) by allele quantification combined with melting curves analysis of single-nucleotide polymorphism loci.

BACKGROUND: Molecular approaches for the detection of chromosomal abnormalities will allow the development of rapid, cost-effective screening strategies. We present here a molecular alternative for the detection of aneuploidies and, more specifically, trisomy 21. METHODS: We used the quantitative value of melting curve analysis of heterozygous genetic loci to establish a relative allelic count. The two alleles of a given single-nucleotide polymorphism (SNP) were differentiated by thermodynamic stability with a fluorescently labeled hybridization probe and were quantified by relative areas of derivative melting curves detected after fluorescence resonance energy transfer. Heterozygous SNPs provided internal controls for the assay. RESULTS: We selected six SNPs, heterozygous in at least 30% of a random population, to form a panel of informative loci in the majority of a random population. After normalization to a heterozygous control, samples segregated into three categories; nontrisomic samples had mean allele ratios of 0.96-1.09, whereas trisomic samples had mean ratios of 1.84-2.09 or 0.46-0.61, depending on which allele was duplicated. Within-run mean CVs of ratios were 6.5-27%, and between-assay mean CVs were 13-24%. CONCLUSIONS: The use of melting curve analysis of multiple SNPs is an alternative to the use of small tandem repeats for the detection of trisomies. Because of the high density of SNPs, the approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome; it is also applicable to aneuploidies other than trisomy 21 and to specimens that are not amenable to cytogenetic analysis.

Alleles↗