Biomedical subjects

D A Nickerson

Publications and source records attributed to D A Nickerson.

At least 19 recordsLinked to original sources

PubMed2000

Localization of a small genomic region associated with elevated ACE.

Defining the relationship between multiple polymorphisms in a small genomic region and an underlying quantitative trait locus (QTL) represents a major challenge in human genetics. Pedigree analyses have shown that angiotensin I-converting enzyme (ACE) levels are influenced by a QTL located within or close to the ACE gene and most likely resides in the 3' region of this locus. We genotyped seven polymorphisms spanning 13 kb in the 3' end of ACE in 159 Afro-Caribbean subjects to evaluate the linkage disequilibrium between these sites and to narrow the genomic region associated with an elevated ACE level using a cladistic analysis. The linkage disequilibrium measurement D' and a haplotype tree revealed three distinct haplotype segments, presumably because of recombination. The value of the linkage disequilibrium parameter p(excess) was highest for site 22982, which is located in the middle segment. A series of nested, cladistic analyses confirmed that the other two regions are unlikely to be the ACE-linked QTL and that the variant resides in the middle region. Analyses of the same polymorphisms in 98 unrelated Europeans in the Monitoring Trends and Determinants in Cardiovascular Diseases (MONICA) study resulted in fewer haplotypes than were observed among the Afro-Caribbean subjects, suggesting that populations with greater genetic diversity may be especially informative for fine-scale mapping.

Black People↗

PubMed2000

Apolipoprotein E variation at the sequence haplotype level: implications for the origin and maintenance of a major human polymorphism.

Three common protein isoforms of apolipoprotein E (apoE), encoded by the epsilon2, epsilon3, and epsilon4 alleles of the APOE gene, differ in their association with cardiovascular and Alzheimer's disease risk. To gain a better understanding of the genetic variation underlying this important polymorphism, we identified sequence haplotype variation in 5.5 kb of genomic DNA encompassing the whole of the APOE locus and adjoining flanking regions in 96 individuals from four populations: blacks from Jackson, MS (n=48 chromosomes), Mayans from Campeche, Mexico (n=48), Finns from North Karelia, Finland (n=48), and non-Hispanic whites from Rochester, MN (n=48). In the region sequenced, 23 sites varied (21 single nucleotide polymorphisms, or SNPs, 1 diallelic indel, and 1 multiallelic indel). The 22 diallelic sites defined 31 distinct haplotypes in the sample. The estimate of nucleotide diversity (site-specific heterozygosity) for the locus was 0.0005+/-0.0003. Sequence analysis of the chimpanzee APOE gene showed that it was most closely related to human epsilon4-type haplotypes, differing from the human consensus sequence at 67 synonymous (54 substitutions and 13 indels) and 9 nonsynonymous fixed positions. The evolutionary history of allelic divergence within humans was inferred from the pattern of haplotype relationships. This analysis suggests that haplotypes defining the epsilon3 and epsilon2 alleles are derived from the ancestral epsilon4s and that the epsilon3 group of haplotypes have increased in frequency, relative to epsilon4s, in the past 200,000 years. Substantial heterogeneity exists within all three classes of sequence haplotypes, and there are important interpopulation differences in the sequence variation underlying the protein isoforms that may be relevant to interpreting conflicting reports of phenotypic associations with variation in the common protein isoforms.

Alleles↗

PubMed2000

Hypertension and single nucleotide polymorphisms.

Hypertension is a common, complex disease phenotype that has been intensively studied to identify susceptibility loci in humans. Candidate genes continue to be uncovered via genetic analysis in model organisms through linkage analysis with families and/or sib-pairs and through association studies using sequence variants in genes that play a role in key pathways regulating blood pressure in humans, such as the renin-angiotensin system (RAS). Recent studies exploring the sequence diversity in human candidate genes suggest that the distribution and organization of single nucleotide polymorphisms (SNPs) within and among human populations is complex. Issues related to the use of SNPs in analyzing the genetic determinants of hypertension are illustrated using recent studies on the angiotensin-converting enzyme (ACE).

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2000

Recombinational and mutational hotspots within the human lipoprotein lipase gene.

Here an analysis is presented of the roles of recombination and mutation in shaping previously determined haplotype variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase gene (LPL), scored in 71 individuals from three populations: 24 African Americans, 24 Finns, and 23 non-Hispanic whites. Recombination and gene-conversion events inferred from data on 88 haplotypes that were defined by 69 variable sites were tested. The analysis revealed 29 statistically significant recombination events and one gene-conversion event. The recombination events were concentrated in a 1.9-kb region, near the middle of the segment, that contains a microsatellite and a pair of tandem and complementary mononucleotide runs; both the microsatellite and the runs show length variation. An analysis of site variation revealed that 9.6% of the nucleotides at CpG sites were variable, as were 3% of the nucleotides found in mononucleotide runs of >/=5 nucleotides, 3% of the nucleotides found </=3 bp from certain putative polymerase alpha-arrest sites, and 0. 5% of the remaining nucleotides. This nonhomogeneous distribution of variation suggests that multiple mutational hits at certain sites are common, an observation that challenges the fundamental assumption of the infinite-sites-mutation model. The nonrandom patterns of recombination and mutation suggest that randomly chosen single-nucleotide polymorphisms may not be optimal for disequilibrium mapping of this gene. Overall, these results indicate that both recombinational and mutational hotspots have played significant roles in shaping the haplotype variation at the LPL locus.

Black People↗

PubMed2000

Cladistic structure within the human Lipoprotein lipase gene and its implications for phenotypic association studies.

Haplotype variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase (LPL) gene was scored in three populations: African-Americans from Jackson, Mississippi (24 individuals), Finns from North Karelia, Finland (24), and non-Hispanic whites from Rochester, Minnesota (23). Earlier analyses had indicated that recombination was common but concentrated into a hotspot and that recurrent mutations at multiple sites may have occurred. We show that much evolutionary structure exists in the haplotype variation on either side of the recombinational hotspot. By peeling off significant recombination events from a tree estimated under the null hypothesis of no recombination, we also reveal some cladistic structure not disrupted by recombination during the time to coalescence of this variation. Additional cladistic structure is estimated to have emerged after recombination. Many apparent multiple mutational events at sites still remain after removing the effects of the detected recombination/gene conversion events. These apparent multiple events are found primarily at sites identified as highly mutable by previous studies, strengthening the conclusion that they are true multiple events. This analysis portrays the complexity of the interplay among many recombinational and mutational events that would be needed to explain the patterns of haplotype diversity in this gene. The cladistic structure in this region is used to identify four to six single-nucleotide polymorphisms (SNPs) that would provide disequilibrium coverage over much of this region. These sites may be useful in identifying phenotypic associations with variable sites in this gene. Evolutionary considerations also imply that the SNPs in the 3' region should have general utility in most human populations, but the 5' SNPs may be more population specific. Choosing SNPs at random would generally not provide adequate disequilibrium coverage of the sequenced region.

Black or African American↗

PubMed2000

Sequence diversity and large-scale typing of SNPs in the human apolipoprotein E gene.

A common strategy for genotyping large samples begins with the characterization of human single nucleotide polymorphisms (SNPs) by sequencing candidate regions in a small sample for SNP discovery. This is usually followed by typing in a large sample those sites observed to vary in a smaller sample. We present results from a systematic investigation of variation at the human apolipoprotein E locus (APOE), as well as the evaluation of the two-tiered sampling strategy based on these data. We sequenced 5.5 kb spanning the entire APOE genomic region in a core sample of 72 individuals, including 24 each of African-Americans from Jackson, Mississippi; European-Americans from Rochester, Minnesota; and Europeans from North Karelia, Finland. This sequence survey detected 21 SNPs and 1 multiallelic indel, 14 of which had not been previously reported. Alleles varied in relative frequency among the populations, and 10 sites were polymorphic in only a single population sample. Oligonucleotide ligation assays (OLA) were developed for 20 of these sites (omitting the indel and a closely-linked SNP). These were then scored in 2179 individuals sampled from the same three populations (n = 843, 884, and 452, respectively). Relative allele frequencies were generally consistent with estimates from the core sample, although variation was found in some populations in the larger sample at SNPs that were monomorphic in the corresponding smaller core sample. Site variation in the larger samples showed no systematic deviation from Hardy-Weinberg expectation. The large OLA sample clearly showed that variation in many, but not all, of OLA-typed SNPs is significantly correlated with the classical protein-coding variants, implying that there may be important substructure within the classical epsilon 2, epsilon 3, and epsilon 4 alleles. Comparison of the levels and patterns of polymorphism in the core samples with those estimated for the OLA-typed samples shows how nucleotide diversity is underestimated when only a subset of sites are typed and underscores the importance of adequate population sampling at the polymorphism discovery stage. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF261279.]

Alleles↗

PubMed1999

Sequence variation in the human angiotensin converting enzyme.

Angiotensin converting enzyme (encoded by the gene DCP1, also known as ACE) catalyses the conversion of angiotensin I to the physiologically active peptide angiotensin II, which controls fluid-electrolyte balance and systemic blood pressure. Because of its key function in the renin-angiotensin system, many association studies have been performed with DCP1. Nearly all studies have associated the presence (insertion, I) or absence (deletion, D) of a 287-bp Alu repeat element in intron 16 with the levels of circulating enzyme or cardiovascular pathophysiologies. Many epidemiological studies suggest that the DCP1*D allele confers increased susceptibility to cardiovascular disease; however, other reports have found no such association or even a beneficial effect. We present here the complete genomic sequence of DCP1 from 11 individuals, representing the longest contiguous scan (24 kb) for sequence variation in human DNA. We identified 78 varying sites in 22 chromosomes that resolved into 13 distinct haplotypes. Of the variant sites, 17 were in absolute linkage disequilibrium with the commonly typed Alu insertion/deletion polymorphism, producing two distinct and distantly related clades. We also identified a major subdivision in the Alu deletion clade that enables further analysis of the traits associated with this gene. The diversity uncovered in DCP1 is comparable to that described for other regions in the human genome. The highly correlated structure in DCP1 raises important issues for the determination of functional DNA variants within genes and genetic studies in humans based on marker association.

Alu Elements↗

PubMed1999

Identification of candidate coding region single nucleotide polymorphisms in 165 human genes using assembled expressed sequence tags.

Using assembled expressed sequence tags (ESTs) from 50 different cDNA libraries, we have identified contigs that represent the complete coding sequences of 850 known human genes, and have scanned these for high quality sequence substitutions. We report the identification and characteristics of 201 candidate single nucleotide polymorphisms found in the coding sequences (cSNPs) of 165 of these genes. Using a conservative calculation, coding region nucleotide diversity (the average number of differences between any pair of chromosomes) was found to be 3 per 10,000 bp based on this data. This analysis reveals that assembled ESTs from multiple libraries may provide a rich source of comparative sequences to search for cSNPs in the human genome.

Amino Acid Substitution↗

PubMed1999

Mining SNPs from EST databases.

There is considerable interest in the discovery and characterization of single nucleotide polymorphisms (SNPs) to enable the analysis of the potential relationships between human genotype and phenotype. Here we present a strategy that permits the rapid discovery of SNPs from publicly available expressed sequence tag (EST) databases. From a set of ESTs derived from 19 different cDNA libraries, we assembled 300,000 distinct sequences and identified 850 mismatches from contiguous EST data sets (candidate SNP sites), without de novo sequencing. Through a polymerase-mediated, single-base, primer extension technique, Genetic Bit Analysis (GBA), we confirmed the presence of a subset of these candidate SNP sites and have estimated the allele frequencies in three human populations with different ethnic origins. Altogether, our approach provides a basis for rapid and efficient regional and genome-wide SNP discovery using data assembled from sequences from different libraries of cDNAs.

DNA, Complementary↗

PubMed1998

Mapping a protein-binding site on straightened DNA by atomic force microscopy.

We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (> 5 kb) at nanometer resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface. The immobilized molecules were detected by AFM. The straightened DNA displayed a sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein-binding site. Using GAL4, a yeast transcription factor, we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site. The technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-molecule level.

Aluminum Silicates↗

PubMed1998

Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome.

Diagnostic re-sequencing plays a central role in medical and evolutionary genetics. In this report we describe a process that applies fluorescence-based re-sequencing and an integrated set of analysis tools to automate and simplify the identification of DNA variations using the human mitochondrial genome as a model system. Two programs used in genome sequence analysis (Phred, a base-caller, and Phrap, a sequence assembler) are applied to assess the quality of each base call across the sequence. Potential DNA variants are automatically identified and 'tagged' by comparing the assembled sequence with a reference sequence. We also show that employing the Consed program to display a set of highly annotated reference sequences greatly simplifies data analysis by providing a visual database containing information on the location of the PCR primers, coding and regulatory sequences and previously known DNA variants. Among the 12 genomes sequenced 378 variants including 29 new variants were identified along with two heteroplasmic sites, automatically detected by the PolyPhred program. Overall we document the ease and speed of performing high quality and accurate fluorescence-based re-sequencing on long tracts of DNA as well as the application of new approaches to automatically find and view DNA variants among these sequences.

Base Sequence↗

PubMed1998

DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene.

Lipoprotein lipase plays a central role in lipid metabolism and the gene that encodes this enzyme (LPL) is a candidate susceptibility gene for cardiovascular disease. Here we report the complete sequence of a fraction of the LPL gene for 71 individuals (142 chromosomes) from three populations that may have different histories affecting the organization of the sequence variation. Eighty-eight sites in this 9.7 kb vary among individuals from these three populations. Of these, 79 were single nucleotide substitutions and 9 sites involved insertion-deletion variations. The average nucleotide diversity across the region was 0.2% (or on average 1 variable site every 500 bp). At 34 of these sites, the variation was found in only one of the populations, reflecting the differing population and mutational histories. If LPL is a typical human gene, the pattern of sequence variation that exists in introns as well as exons, even for the small number of samples considered here, will present challenges for the identification of sites, or combinations of sites, that influence variation in risk of disease in the population at large.

Base Sequence↗

PubMed1998

Haplotype structure and population genetic inferences from nucleotide-sequence variation in human lipoprotein lipase.

Allelic variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase gene (LPL) was scored in 71 healthy individuals (142 chromosomes) from three populations: African Americans (24) from Jackson, MS; Finns (24) from North Karelia, Finland; and non-Hispanic Whites (23) from Rochester, MN. The sequences had a total of 88 variable sites, with a nucleotide diversity (site-specific heterozygosity) of .002+/-.001 across this 9.7-kb region. The frequency spectrum of nucleotide variation exhibited a slight excess of heterozygosity, but, in general, the data fit expectations of the infinite-sites model of mutation and genetic drift. Allele-specific PCR helped resolve linkage phases, and a total of 88 distinct haplotypes were identified. For 1,410 (64%) of the 2,211 site pairs, all four possible gametes were present in these haplotypes, reflecting a rich history of past recombination. Despite the strong evidence for recombination, extensive linkage disequilibrium was observed. The number of haplotypes generally is much greater than the number expected under the infinite-sites model, but there was sufficient multisite linkage disequilibrium to reveal two major clades, which appear to be very old. Variation in this region of LPL may depart from the variation expected under a simple, neutral model, owing to complex historical patterns of population founding, drift, selection, and recombination. These data suggest that the design and interpretation of disease-association studies may not be as straightforward as often is assumed.

Animals↗

PubMed1998

Oligonucleotide ligation assay for detecting mutations in the human immunodeficiency virus type 1 pol gene that are associated with resistance to zidovudine, didanosine, and lamivudine.

This report describes the detection of mutations in the pol gene of human immunodeficiency virus type 1 associated with resistance to zidovudine, didanosine, and lamivudine by genotyping by an oligonucleotide ligation assay specific codons in the pol gene amplified by PCR. Our studies demonstrate the sensitivity, simplicity, and specificity of this genotyping system.

Anti-HIV Agents↗

PubMed1997

PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing.

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.

Animals↗

PubMed1996

Single-well genotyping of diallelic sequence variations by a two-color ELISA-based oligonucleotide ligation assay.

Single nucleotide substitutions and unique insertions/deletions are the most common form of DNA sequence variation and disease-causing mutation in the human genome. Because of the biological and medical importance of these variations, a wide array of methods have been developed for their typing. We have applied an approach that combines the amplification of polymorphic regions by the polymerase chain reaction (PCR) with a system for typing diallelic variants using an oligonucleotide ligation assay (OLA). In this report, we describe a significant advance in this technology that permits the typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors. We demonstrate the specificity, sensitivity and ease of data interpretation with this system. Furthermore, we show that multiplex PCR/OLA not only increases the throughput of DNA typing but also increases its accuracy in typing diallelic sequence variations using an approach that can be broadly applied for human genome analysis (in evaluating genotype/phenotype links), in typing infectious agents and in forensic analysis.

Alleles↗

PubMed1996

Increasing the information content of STS-based genome maps: identifying polymorphisms in mapped STSs.

Physical maps of the human genome are being constructed by many groups using a mapping strategy that relies on the development of sequence-tagged sites (STSs). Thousands of physically mapped STSs, representing hundreds of kilobases (kb) of unique human DNA sequence, have been generated by these efforts. Since sequence variations are found every 1-2 kb in the genome, it is possible to extract additional information from mapped STSs by scanning them for variations. By screening 154 of the STSs published by the Whitehead Institute/MIT Genome Center, we have identified 47 new DNA sequence polymorphisms among the 37.2 kb of unique DNA sequence contained in these STSs. Using a sequence-based approach to estimate allele frequencies for these variations, 29 of the substitution polymorphisms (1 in 1.3 kb) were found to have heterozygosities exceeding 32%. Our study shows that the information content of STS-based genome maps can be increased with minimal additional effort by scanning for DNA polymorphisms, and that ambiguities and errors in the initial STS sequence can be resolved and corrected in the process.

Alleles↗

PubMed1996

AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns.

Taq DNA polymerases in which the phenylalanine is substituted by a tyrosine at position 667 (Taq F667Y) are members of a new class of DNA polymerases that incorporate chain-terminating dideoxyribonucleoside triphosphates (ddNTPs) much more efficiently than the wild-type Taq DNA polymerase. Improved incorporation of ddNTPs into DNA during cycle sequencing using AmpliTaq DNA polymerase, FS (Taq-FS, a member of the Taq F667Y family), and dye-labeled primers results in nearly uniform peak heights in the sequencing trace. This is not the case when dye-labeled ddNTPs are used in Taq-FS cycle sequencing reactions. While the rate of dye-terminator incorporation is more efficient with Taq-FS, the peak pattern is still highly variable and different from that produced by the wild-type enzyme. We have systematically examined pairs of sequence-tagged sites that vary at only a single nucleotide to determine how base changes influence the peak heights of neighboring bases in sequencing traces generated by the Taq-FS dye-terminator chemistry. In 31 of 64 possible 3-base windows (48%), we find that the peak height of a particular base can be predicted by knowing just one or two bases 5' to the base in question. We have also compared and contrasted the peak patterns produced by the Taq-FS enzyme with those previously identified for the wild-type enzyme. Establishing the patterns in peak heights within local sequence contexts can improve the accuracy of base-calling and the identification of polymorphisms/mutations when using the Taq-FS dye-terminator cycle-sequencing chemistry.

DNA-Directed DNA Polymerase↗