Alkaline-mediated differential interaction (AMDI): A simple automatable single-nucleotide polymorphism assay

The key requirements for high-throughput single-nucleotide polymorphism (SNP) typing of DNA samples in large-scale disease case-control studies are automatability, simplicity, and robustness, coupled with minimal cost. In this paper we describe a fluorescence technique for the detection of SNPs that have been amplified by using the amplification refractory mutation system (ARMS)-PCR procedure. Its performance was evaluated using 32 sequence-specific primer mixes to assign the HLA-DRB alleles to 80 lymphoblastoid cell line DNAs chosen from our database for their diversity. All had been typed previously by alternative methods, either direct sequencing or gel electrophoresis. We believe the detection system that we call AMDI (alkaline-mediated differential interaction) satisfies the above criteria and is suitable for general high-throughput SNP typing.

A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers

BRCA1 and BRCA2 carriers are at increased risk for both breast and ovarian cancer, but estimates of lifetime risk vary widely, suggesting their penetrance is modified by other genetic and/or environmental factors. The BRCA1 and BRCA2 proteins function in DNA repair in conjunction with RAD51. A preliminary report suggested that a single nucleotide polymorphism in the 5′ untranslated region of RAD51 (135C/G) increases breast cancer risk in BRCA1 and BRCA2 carriers. To investigate this effect we studied 257 female Ashkenazi Jewish carriers of one of the common BRCA1 (185delAG, 5382insC) or BRCA2 (6174delT) mutations. Of this group, 164 were affected with breast and/or ovarian cancer and 93 were unaffected. RAD51 genotyping was performed on all subjects. Among BRCA1 carriers, RAD51-135C frequency was similar in healthy and affected women [6.1% (3 of 49) and 9.9% (12 of 121), respectively], and RAD-135C did not influence age of cancer diagnosis [Hazard ratio (HR) = 1.18 for disease in RAD51-135C heterozygotes, not significant]. However, in BRCA2 carriers, RAD51-135C heterozygote frequency in affected women was 17.4% (8 of 46) compared with 4.9% (2 of 41) in unaffected women (P = 0.07). Survival analysis in BRCA2 carriers showed RAD51-135C increased risk of breast and/or ovarian cancer with an HR of 4.0 [95% confidence interval 1.6–9.8, P = 0.003]. This effect was largely due to increased breast cancer risk with an HR of 3.46 (95% confidence interval 1.3–9.2, P = 0.01) for breast cancer in BRCA2 carriers who were RAD51-135C heterozygotes. RAD51 status did not affect ovarian cancer risk. These results show RAD51-135C is a clinically significant modifier of BRCA2 penetrance, specifically in raising breast cancer risk at younger ages.

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP–Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.

Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3́end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.

Operational RNA code for amino acids: species-specific aminoacylation of minihelices switched by a single nucleotide.

The genetic code is based on aminoacylation reactions where specific amino acids are attached to tRNAs bearing anticodon trinucleotides. However, the anticodon-independent specific aminoacylation of RNA minihelix substrates by bacterial and yeast tRNA synthetases suggested an operational RNA code for amino acids whereby specific RNA sequences/structures in tRNA acceptor stems correspond to specific amino acids. Because of the possible significance of the operational RNA code for the development of the genetic code, we investigated aminoacylation of synthetic RNA minihelices with a human enzyme to understand the sequences needed for that aminoacylation compared with those needed for a microbial system. We show here that the species-specific aminoacylation of glycine tRNAs is recapitulated by a species-specific aminoacylation of minihelices. Although the mammalian and Escherichia coli minihelices differ at 6 of 12 base pairs, two of the three nucleotides essential for aminoacylation by the E. coli enzyme are conserved in the mammalian minihelix. The two conserved nucleotides were shown to be also important for aminoacylation of the mammalian minihelix by the human enzyme. A simple interchange of the differing nucleotide enabled the human enzyme to now charge the bacterial substrate and not the mammalian minihelix. Conversely, this interchange made the bacterial enzyme specific for the mammalian substrate. Thus, the positional locations (if not the actual nucleotides) for the operational RNA code for glycine appear conserved from bacteria to mammals.

Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs.

The Genetics of Success: How Single-Nucleotide Polymorphisms Associated With Educational Attainment Relate to Life-Course Development.

A previous genome-wide association study (GWAS) of more than 100,000 individuals identified molecular-genetic predictors of educational attainment. We undertook in-depth life-course investigation of the polygenic score derived from this GWAS using the four-decade Dunedin Study (N = 918). There were five main findings. First, polygenic scores predicted adult economic outcomes even after accounting for educational attainments. Second, genes and environments were correlated: Children with higher polygenic scores were born into better-off homes. Third, children's polygenic scores predicted their adult outcomes even when analyses accounted for their social-class origins; social-mobility analysis showed that children with higher polygenic scores were more upwardly mobile than children with lower scores. Fourth, polygenic scores predicted behavior across the life course, from early acquisition of speech and reading skills through geographic mobility and mate choice and on to financial planning for retirement. Fifth, polygenic-score associations were mediated by psychological characteristics, including intelligence, self-control, and interpersonal skill. Effect sizes were small. Factors connecting DNA sequence with life outcomes may provide targets for interventions to promote population-wide positive development.

Development of single nucleotide polymorphism (SNP) markers from the mango (Mangifera indica) transcriptome for mapping and estimation of genetic diversity

The development of molecular markers for genomic studies in Mangifera indica (mango) will allow marker-assisted selection and identification of genetically diverse germplasm, greatly aiding mango breeding programs. We report here our identification of thousands of unambiguous molecular markers that can be easily assayed across genotypes of the species. With origin centered in Southeast Asia, mangos are grown throughout the tropics and subtropics as a nutritious fruit that exhibits remarkable intraspecific phenotypic diversity. With the goal of building a high density genetic map, we have undertaken discovery of sequence variation in expressed genes across a broad range of mango cultivars. A transcriptome sequence reference was built de novo from extensive sequencing and assembly of RNA from cultivar 'Tommy Atkins'. Single nucleotide polymorphisms (SNPs) in protein coding transcripts were determined from alignment of RNA reads from 24 mango cultivars of diverse origins: 'Amin Abrahimpur' (India), 'Aroemanis' (Indonesia), 'Burma' (Burma), 'CAC' (Hawaii), 'Duncan' (Florida), 'Edward' (Florida), 'Everbearing' (Florida), 'Gary' (Florida), 'Hodson' (Florida), 'Itamaraca' (Brazil), 'Jakarata' (Florida), 'Long' (Jamaica), 'M. Casturi Purple' (Borneo), 'Malindi' (Kenya), 'Mulgoba' (India), 'Neelum' (India), 'Peach' (unknown), 'Prieto' (Cuba), 'Sandersha' (India), 'Tete Nene' (Puerto Rico), 'Thai Everbearing' (Thailand), 'Toledo' (Cuba), 'Tommy Atkins' (Florida) and 'Turpentine' (West Indies). SNPs in a selected subset of protein coding transcripts are currently being converted into Fluidigm assays for genotyping of mapping populations and germplasm collections. Using an alternate approach, SNPs (144) discovered by sequencing of candidate genes in 'Kensington Pride' have already been converted and used for genotyping.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in the rpoB, katG, inhA, ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 for rpoB, katG, inhA, ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.