Molecular Genetics

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Now showing 1 - 5 of 5
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    A Non-Parametric Alternative to The Cochran-Armitage Trend Test in Genetic Case-Control Association Studies: The Two-sided Jonckheere's Test
    (2020) Zhou, Zhengyang; Ku, Hung-Chih; Xing, Chao; Manning, Sydney E.
    Purpose: In genetic association studies with case-control design, standard practice is to perform the Cochran-Armitage (CA) trend test under the assumption of additive genetic model. The CA trend test is a parametric statistical test, and under the null hypothesis of no association between the genetic variant and disease, the test statistic asymptotically follows a chi-square distribution with 1 degree-of-freedom. However, when the sample size and/or variant minor allele frequency are small, asymptotic properties may not hold, which can lead to reduced statistical power in detecting genetic associations. Methods: To improve statistical power in this case, we consider the two-sided Jonckheere's test, which is a rank-based nonparametric test. By not imposing assumptions on the distributions of the data, it is expected to have better statistical power than parametric tests for small sample sizes and/or rare variants. We conducted extensive simulations to compare the statistical power between the CA trend test and the two-sided Jonckheere's test under various scenarios. Results: We found for small sample size (e.g., n=200) and low minor allele frequency (e.g., p=0.05), the two-sided Jonckheere's test outpowered the CA trend test for all genetic models ranging from recessive to dominant. Conclusion: This finding provides an alternative to the CA trend test in genetic association studies under these circumstances. With higher statistical power from the two-sided Jonckheere's test, genetic epidemiologists will be able to detect more genetic associations for complex diseases, which may lead to better prevention and treatment strategies.
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    Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification
    (2020) Phillips, Nicole; Planz, John; Johnson, Gretchen A.
    Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins for oxidative phosphorylation. Depending on the cell type and stage of development, each cell contains an average of 10^3 to 10^4 copies of mtDNA. Current methods of mtDNA copy number quantification can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both components is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate ratio estimates. Methods: We employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus (β2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This assay was optimized so both targets amplify with similar efficiency to give precise and accurate ratio estimates. The precision was demonstrated over repeated runs and shown to be accurate based on SRM quantification. Conclusion: This protocol demonstrates reproducible quantification in both mitochondrial and nuclear targets while reducing time and resources. The data generated can be used to indicate overall mitochondrial health and can be utilized in research areas such as aging, cancer, forensics and neurodevelopment.
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    Effect of Functional Polymorphisms in ABCB1 gene on Opioid Doses in Patients with Chronic Low Back Pain
    (2020) Lin, Annie
    Purpose Opioids are commonly used to treat pain. P-glycoprotein (P-GP) is encoded by the ABCB1 gene and acts as an efflux pump at the blood-brain barrier and reduces the intracellular penetration of opioids. Research suggests single nucleotide polymorphisms (SNP) C3435T and G2677T/A in the ABCB1 gene are associated with higher levels of P-GP expression. We hypothesize that the presence of a CC-GG diplotype will affect opioid doses in patients with chronic low back pain. Methods A total of 71 patients from the PRECISION Pain Research Registry participated in this study. All patients met the NIH criteria for chronic back pain and reported a current use of opioids. Patients were genotyped for the C3435T and G2677T/A SNPs with the Illumina Infinium Global Screening Array. Patient reported drug data was calculated into morphine milligram equivalents (MMEs). Results The mean MMEs for patients with the CC and CT/TT genotypes were 30.4 and 31.0, respectively (p=0.73). The mean MMEs for patients with the GG and GT/TT genotypes were 28.58 and 34.37, respectively (p=0.43). Mean MMEs for a CC-GG diplotype vs other diplotypes were 29.6 and 32.9, respectively (p=0.67). Adjustment for baseline age, gender, pain intensity, pain catastrophizing and pain self-efficacy did not materially affect the observed results. Conclusions We were unable to detect a significant association between these ABCB1 variants and MME doses. This may be potentially attributed to the limited statistical power of this study.
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    The genomic architecture of the latent variable δ homolog (dEQ) in Mexican Americans and non-Hispanic whites
    (2020) Phillips, Nicole; Silzer, Talisa; Barber, Robert; Royall, Don; Palmer, Raymond; Colmenarez, Micaela
    The latent variable δ homolog (dEQ) has been established as a reliable indicator of cognitive performance and a predictor of future dementia. Here, we sought to identify genetic variants underlying dEQ in both non-Hispanic White (NWH) and Mexican American (MA) populations, hypothesizing that novel genomic risk loci for dementia will be implicated in the MA cohort. Genotyping was performed on the TARCC cohort using the Illumina® MEGA Array, which includes ~1.7 million SNPs. dEQ was generated by Donald Royall and Ray Palmer (UT Health Science Center @ San Antonio) for the entire genotyped cohort (nNHW= 1572; nMA=1030). Association testing was conducted using PLINK (Purcell et al., 2007) according to standard procedures (Anderson et al., 2010). The primary genetic association with dEQ in NHW is the TOMM40/APOE locus (rs4420638, p=9.477x10-32); the association for this locus in the MA cohort was much less significant (p=3.886x10-5). Among MAs, three interesting and unique loci emerged as suggestive: XIRP2 (chr2, rs7595556 p=8.165x10-6), KIF13A (chr6, rs7766167, p= 3.404x10-6), and LINC00907 (chr18, rs237972, p=7.88x10-7). The loci discovered in the MA cohort have not previously been implicated in dementia/Alzheimer's disease; however, variants in these genes have been associated with metabolic phenotypes that are particularly relevant to the pathophysiology of cognitive decline in MAs: diabetic neuropathy, epigenetic aging, and childhood obesity. Further investigation of these genomic regions may illuminate mechanisms by which metabolic syndromes may confer risk for earlier onset of age-related cognitive decline in MAs.
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    Taking the bait: A PCR-free enrichment strategy for nanopore sequencing applications
    (2020) Planz, John V.; Sedlazeck, Fritz; Zascavage, Roxanne R.; Hall, Courtney
    Purpose: DNA methylation is a critical epigenetic modification involved in regulating gene expression. Although aberrant cytosine methylation has been implicated in diseases ranging from neurological disorders to cancer, current understanding is obscured by the inherent limitations of bisulfite conversion. Nanopore sequencing offers the ability to simultaneously ascertain genetic variation and base modifications without chemical treatment. While numerous copies of target regions may be present within a sample, this represents a small fraction of total genetic material competing for pore access. Here we evaluate RNA bait hybridization capture for enrichment of mitochondrial DNA (mtDNA) prior to nanopore sequencing. Methods: Heavy and light strands were individually captured using the Arbor Biosciences myBaits Mito panel. Elutant from the first capture served as rebaiting input with the opposite probe set. Double-stranded products subsequently generated were multiplexed and sequenced on the MinION device. Resultant data were separated by barcode and mapped to the human reference genome. Read counts were normalized for coverage comparisons and methylation was detected using Tombo. Results: Overall input and throughput were significantly lower than a typical whole genome sequencing run, however mtDNA read counts indicate successful enrichment. In addition to ensuring target regions outcompete background DNA, these techniques allowed for methylation detection within native strands. Conclusion: The significance of a PCR-free enrichment strategy for nanopore sequencing applications extends beyond mtDNA. These techniques have been used to capture challenging regions within the rat genome and could provide novel insights into the genetic and epigenetic landscape of other biomedically relevant regions.