Human Single Nucleotide Polymorphisms Research Articles

The sequences of 150,119 genomes in the UK Biobank

Detailed knowledge of how diversity in the sequence of the human genome affects phenotypic diversity depends on a comprehensive and reliable characterization of both sequences and phenotypic variation. Over the past decade, insights into this relationship have been obtained from whole-exome sequencing or whole-genome sequencing of large cohorts with rich phenotypic data1,2. Here we describe the analysis of whole-genome sequencing of 150,119 individuals from the UK Biobank3. This constitutes a set of high-quality variants, including 585,040,410 single-nucleotide polymorphisms, representing 7.0% of all possible human single-nucleotide polymorphisms, and 58,707,036 indels. This large set of variants allows us to characterize selection based on sequence variation within a population through a depletion rank score of windows along the genome. Depletion rank analysis shows that coding exons represent a small fraction of regions in the genome subject to strong sequence conservation. We define three cohorts within the UK Biobank: a large British Irish cohort, a smaller African cohort and a South Asian cohort. A haplotype reference panel is provided that allows reliable imputation of most variants carried by three or more sequenced individuals. We identified 895,055 structural variants and 2,536,688 microsatellites, groups of variants typically excluded from large-scale whole-genome sequencing studies. Using this formidable new resource, we provide several examples of trait associations for rare variants with large effects not found previously through studies based on whole-exome sequencing and/or imputation.

New Frontiers in Neurodegeneration and Regeneration Associated with Brain-Derived Neurotrophic Factor and the rs6265 Single Nucleotide Polymorphism.

Brain-derived neurotrophic factor is an extensively studied neurotrophin implicated in the pathology of multiple neurodegenerative and psychiatric disorders including, but not limited to, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, traumatic brain injury, major de-pressive disorder, and schizophrenia. Here we provide a brief summary of current knowledge on the role of BDNF and the common human single nucleotide polymorphism, rs6265, in driving the pathogenesis and rehabilitation in these disorders, as well as the status of BDNF-targeted therapies. A common trend has emerged correlating low BDNF levels, either detected within the central nervous system or peripherally, to disease states, suggesting that BDNF replacement therapies may hold clinical promise. In addition, we introduce evidence for a distinct role of the BDNF pro-peptide as a biologically active ligand and the need for continuing studies on its neurological function outside of that as a molecular chaperone. Finally, we highlight the latest research describing the role of rs6265 expression in mechanisms of neurodegeneration as well as paradoxical advances in the understanding of this genetic variant in neuroregeneration. All of this is discussed in the context of personalized medicine, acknowledging there is no “one size fits all” therapy for neurodegenerative or psychiatric disorders and that continued study of the multiple BDNF isoforms and genetic variants represents an avenue for discovery ripe with therapeutic potential.

A Common Human Brain-Derived Neurotrophic Factor Polymorphism Leads to Prolonged Depression of Excitatory Synaptic Transmission by Isoflurane in Hippocampal Cultures.

Multiple presynaptic and postsynaptic targets have been identified for the reversible neurophysiological effects of general anesthetics on synaptic transmission and neuronal excitability. However, the synaptic mechanisms involved in persistent depression of synaptic transmission resulting in more prolonged neurological dysfunction following anesthesia are less clear. Here, we show that brain-derived neurotrophic factor (BDNF), a growth factor implicated in synaptic plasticity and dysfunction, enhances glutamate synaptic vesicle exocytosis, and that attenuation of vesicular BDNF release by isoflurane contributes to transient depression of excitatory synaptic transmission in mice. This reduction in synaptic vesicle exocytosis by isoflurane was acutely irreversible in neurons that release less endogenous BDNF due to a polymorphism (BDNF Val66Met; rs6265) compared to neurons from wild-type mice. These effects were prevented by exogenous application of BDNF. Our findings identify a role for a common human BDNF single nucleotide polymorphism in persistent changes of synaptic function following isoflurane exposure. These short-term persistent alterations in excitatory synaptic transmission indicate a role for human genetic variation in anesthetic effects on synaptic plasticity and neurocognitive function.

Key amino acids alter activity and trafficking of a well-conserved olfactory receptor.

In this study, we elucidate factors that regulate the trafficking and activity of a well-conserved olfactory receptor (OR), olfactory receptor 558 (Olfr558), and its human ortholog olfactory receptor 51E1 (OR51E1). Results indicate that butyrate activates Olfr558/OR51E1 leading to the production of cAMP, and evokes Ca2+ influx. We also find olfactory G protein (Golf) increases cAMP production induced by Olfr558/OR51E1 activation but does not affect trafficking. Given the 93% sequence identity between OR51E1 and Olfr558, it is surprising to note that OR51E1 has significantly more surface expression yet similar total protein expression. We find that replacing the Olfr558 N-terminus with that of OR51E1 significantly increases trafficking; in contrast, there is no change in surface expression conferred by the OR51E1 TM2, TM3, or TM4 domains. A previous analysis of human OR51E1 single nucleotide polymorphisms (SNPs) identified an A156T mutant primarily found in South Asia as the most abundant (albeit still rare). We find that the OR51E1 A156T mutant has reduced surface expression and cAMP production without a change in total protein expression. In sum, this study of a well-conserved olfactory receptor identifies both protein regions and specific amino acid residues that play key roles in protein trafficking and also elucidates common effects of Golf on the regulation of both the human and murine OR.

Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences

CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases.

Extracellular superoxide dismutase (EC-SOD) R213G variant reduces mitochondrial ROS and preserves mitochondrial function in bleomycin-induced lung injury: EC-SOD R213G variant and intracellular redox regulation

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the lung and vasculature. A common human single nucleotide polymorphism (SNP) in the matrix binding region of EC-SOD leads to a single amino acid substitution, R213G, and alters EC-SOD tissue binding affinity. The change in tissue binding affinity redistributes EC-SOD from tissue to extracellular fluids. Mice (R213G mice) expressing a knock-in of this EC-SOD SNP exhibit elevated plasma and reduced lung EC-SOD content and activity and are protected against bleomycin-induced lung injury and inflammation. It is unknown how the redistribution of EC-SOD alters site-specific redox-regulated molecules relevant for protection. In this study, we tested the hypothesis that the change in the local EC-SOD content would influence not only the extracellular redox microenvironment where EC-SOD is localized but also protect the intracellular redox status of the lung. Mice were treated with bleomycin and harvested 7 days post-treatment. Superoxide levels, measured by electron paramagnetic resonance (EPR), were lower in plasma and Bronchoalveolar lavage fluid (BALF) cells in R213G mice compared to wild-type (WT) mice, while lung cellular superoxide levels in R213G mice were not elevated post-bleomycin compared to WT mice despite low lung EC-SOD levels. Lung glutathione redox potential (EhGSSG), determined by HPLC and fluorescence, was more oxidized in WT compared to R213G mice. In R213G mice, lung mitochondrial oxidative stress was reduced shown by mitochondrial superoxide level measured by EPR in lung and the resistance to bleomycin-induced cardiolipin oxidation. Bleomycin treatment suppressed mitochondrial respiration in WT mice. Mitochondrial function was impaired at baseline in R213G mice but did not exhibit further suppression in respiration post-bleomycin. Collectively, the results indicate that R213G variant preserves intracellular redox state and protects mitochondrial function in the setting of bleomycin-induced inflammation.

Deficiency of transmembrane AMPA receptor regulatory protein γ-8 leads to attention-deficit hyperactivity disorder-like behavior in mice.

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder prevalent in school-age children. At present, however, its etiologies and risk factors are unknown. Transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8 (TARP γ-8, also known as calcium voltage-gated channel auxiliary subunit gamma 8 (CACNG8)) is an auxiliary AMPA receptor (AMPAR) subunit. Here, we report an association between TARP γ-8 and ADHD, whereby adolescent TARP γ-8 knockout (KO) mice exhibited ADHD-like behaviors, including hyperactivity, impulsivity, anxiety, impaired cognition, and memory deficits. Human single-nucleotide polymorphism (SNP) analysis also revealed strong associations between intronic alleles in CACNG8 genes and ADHD susceptibility. In addition, synaptosomal proteomic analysis revealed dysfunction of the AMPA glutamate receptor complex in the hippocampi of TARP γ-8 KO mice. Proteomic analysis also revealed dysregulation of dopaminergic and glutamatergic transmissions in the prefrontal cortices of TARP γ-8 KO mice. Methylphenidate (MPH), which is commonly used to treat ADHD, significantly rescued the major behavioral deficits and abnormal synaptosomal proteins in TARP γ-8 KO mice. Notably, MPH significantly reversed the up-regulation of Grik2 and Slc6a3 in the prefrontal cortex. MPH also significantly improved synaptic AMPAR complex function by up-regulating other AMPAR auxiliary proteins in hippocampal synaptosomes. Taken together, our results suggest that TARP γ-8 is involved in the development of ADHD in humans. This study provides a useful alternative animal model with ADHD-like phenotypes related to TARP γ-8 deficiency, which has great potential for the development of new therapies.

Increased Diagnostic Yield of Array Comparative Genomic Hybridization for Autism Spectrum Disorder in One Institution in Taiwan.

Background and Objectives: Chromosomal microarray offers superior sensitivity for identification of submicroscopic copy number variants (CNVs) and is recommended for the initial genetic testing of patients with autism spectrum disorder (ASD). This study aims to determine the diagnostic yield of array comparative genomic hybridization (array-CGH) in ASD patients from a cohort of Chinese patients in Taiwan. Materials and Methods: Enrolled in this study were 80 ASD children (49 males and 31 females; 2–16 years old) followed up at Taipei MacKay Memorial Hospital between January 2010 and December 2020. The genomic DNA extracted from blood samples was analyzed by array-CGH via the Affymetrix GeneChip Genome-Wide Human single nucleotide polymorphism (SNP) and NimbleGen International Standards for Cytogenomic Arrays (ISCA) Plus Cytogenetic Arrays. The CNVs were classified into five groups: pathogenic (pathologic variant), likely pathogenic (potential pathologic variant), likely benign (potential normal genomic variant), benign (normal genomic variant), and uncertain clinical significance (variance of uncertain significance), according to the American College of Medical Genetics (ACMG) guidelines. Results: We identified 47 CNVs, 31 of which in 27 patients were clinically significant. The overall diagnostic yield was 33.8%. The most frequently clinically significant CNV was 15q11.2 deletion, which was present in 4 (5.0%) patients. Conclusions: In this study, a satisfactory diagnostic yield of array-CGH was demonstrated in a Taiwanese ASD patient cohort, supporting the clinical usefulness of array-CGH as the first-line testing of ASD in Taiwan.

Cross Species use of Human Microarray Genotyping Technology for Bornean Orangutan (Pongo pygmaeus) SNP Discovery

Previous genetic studies of orangutans (Pongo spp.) have relied mainly upon mitochondrial DNA or microsatellite short tandem repeats (STR) for genomic genotyping analysis. Scientists have yet to take advantage of the genetic closeness of the great apes to humans for genomic analysis by using advanced techniques available for human genotyping. To genotype orangutans at Tanjung Puting National Park, we developed a novel combination of a methyl-based magnetic enrichment capture of genomic fecal DNA with genotyping on a human targeted single nucleotide polymorphism (SNP) microarray, and compared this to additional microsatellite (STR) micro-capillary genotyping. We successfully isolated 125 known human genomic SNP loci (0.08% of those targeted) which hybridized orangutan DNA on the human targeted Illumina Infinium QC array. We estimated genetic diversity and relatedness (r) using three estimators for a total of 32 (21 female and 9 male) wild orangutans at the Camp Leakey study site. Average TrioML relatedness within the sample, estimated from our combo SNP/STR dataset, was at a range consistent with half and first cousins (r = .082). All sampled males and females had relatives within the study site indicating we have verified a local, closely related community of wild orangutans at Camp Leakey.

Interaction between M. tuberculosis Lineage and Human Genetic Variants Reveals Novel Pathway Associations with Severity of TB.

Tuberculosis (TB) remains a major public health threat globally, especially in sub-Saharan Africa. Both human and Mycobacterium tuberculosis (MTBC) genetic variation affect TB outcomes, but few studies have examined if and how the two genomes interact to affect disease. We hypothesize that long-term coexistence between human genomes and MTBC lineages modulates disease to affect its severity. We examined this hypothesis in our TB household contact study in Kampala, Uganda, in which we identified three MTBC lineages, of which one, L4.6-Uganda, is clearly derived and hence recent. We quantified TB severity using the Bandim TBscore and examined the interaction between MTBC lineage and human single-nucleotide polymorphisms (SNPs) genome-wide, in two independent cohorts of TB cases (n = 149 and n = 127). We found a significant interaction between an SNP in PPIAP2 and the Uganda lineage (combined p = 4 × 10−8). PPIAP2 is a pseudogene that is highly expressed in immune cells. Pathway and eQTL analyses indicated potential roles between coevolving SNPs and cellular replication and metabolism as well as platelet aggregation and coagulation. This finding provides further evidence that host–pathogen interactions affect clinical presentation differently than host and pathogen genetic variation independently, and that human–MTBC coevolution is likely to explain patterns of disease severity.

Human host genetics and susceptibility to ZIKV infection.

Managing emerging infectious diseases is a current challenge in the fields of microbiology and epidemiology. Indeed, among other environmental and human-related factors, climate change and global warming favor the emergence of new pathogens. The recent Zika virus (ZIKV) epidemic, of which the large and rapid spread surprised the scientific community, is a reminder of the importance to study viruses currently responsible for sporadic infections. Increasing our knowledge of key factors involved in emerging infections is essential to implement specific monitoring that can be oriented according to the pathogen, targeted population, or at-risk environment. Recent technological developments, such as high-throughput sequencing, genome-wide association studies and CRISPR screenings have allowed the identification of human single nucleotide polymorphisms (SNPs) involved in infectious disease outcome. This review focuses on the human genetic host factors that have been identified and shown to be associated with the pathogenesis of ZIKV infection and candidate SNP targets.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational ‘hot-spots’ or ‘cold-spots’. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Genetic architecture associated with familial short stature

Context Human height is an inheritable, polygenic trait under complex and multilocus genetic regulation. Familial short stature (FSS; also called genetic short stature) is the most common type of short stature and is insufficiently known. Objective To investigate the FSS genetic profile and develop a polygenic risk predisposition score for FSS risk prediction. Design and setting The FSS participant group of Han Chinese ancestry was diagnosed by pediatric endocrinologists in Taiwan. Patients and interventions The genetic profiles of 1163 participants with FSS were identified by using a bootstrapping subsampling and genome-wide association studies (GWAS) method. Main outcome measures Genetic profile, polygenic risk predisposition score for risk prediction. Results Ten novel genetic single nucleotide polymorphisms (SNPs) and 9 reported GWAS human height-related SNPs were identified for FSS risk. These 10 novel SNPs served as a polygenic risk predisposition score for FSS risk prediction (area under the curve: 0.940 in the testing group). This FSS polygenic risk predisposition score was also associated with the height reduction regression tendency in the general population. Conclusion A polygenic risk predisposition score composed of 10 genetic SNPs is useful for FSS risk prediction and the height reduction tendency. Thus, it might contribute to FSS risk in the Han Chinese population from Taiwan.

Retraction Note: Detection and replication of epistasis influencing transcription in humans.

Epistasis has rarely been shown among natural polymorphisms in human traits; this research using advanced computation and gene expression data reveals many instances of epistasis between common single nucleotide polymorphisms in humans, with epistasis and the direction of its effect replicating in independent cohorts. Although frequently demonstrated in model organisms and domesticated species, very few examples of epistasis — where the effect of one polymorphism on a trait depends on other polymorphisms present in the genome — have been demonstrated in humans. Using advanced computation and a gene expression study design, these authors reveal hundreds of instances of epistasis between common single nucleotide polymorphisms (SNPs) in humans. They show that epistasis and direction of its effect replicate in independent cohorts. Epistatic networks of three or more SNPs are shown to influence the expression levels of many genes, whereby one cis-acting SNP is modulated by several trans-acting SNPs. Epistasis is the phenomenon whereby one polymorphism’s effect on a trait depends on other polymorphisms present in the genome. The extent to which epistasis influences complex traits1 and contributes to their variation2,3 is a fundamental question in evolution and human genetics. Although often demonstrated in artificial gene manipulation studies in model organisms4,5, and some examples have been reported in other species6, few examples exist for epistasis among natural polymorphisms in human traits7,8. Its absence from empirical findings may simply be due to low incidence in the genetic control of complex traits2,3, but an alternative view is that it has previously been too technically challenging to detect owing to statistical and computational issues9. Here we show, using advanced computation10 and a gene expression study design, that many instances of epistasis are found between common single nucleotide polymorphisms (SNPs). In a cohort of 846 individuals with 7,339 gene expression levels measured in peripheral blood, we found 501 significant pairwise interactions between common SNPs influencing the expression of 238 genes (P < 2.91 × 10−16). Replication of these interactions in two independent data sets11,12 showed both concordance of direction of epistatic effects (P = 5.56 × 10−31) and enrichment of interaction P values, with 30 being significant at a conservative threshold of P < 9.98 × 10−5. Forty-four of the genetic interactions are located within 5 megabases of regions of known physical chromosome interactions13 (P = 1.8 × 10−10). Epistatic networks of three SNPs or more influence the expression levels of 129 genes, whereby one cis-acting SNP is modulated by several trans-acting SNPs. For example, MBNL1 is influenced by an additive effect at rs13069559, which itself is masked by trans-SNPs on 14 different chromosomes, with nearly identical genotype–phenotype maps for each cis–trans interaction. This study presents the first evidence, to our knowledge, for many instances of segregating common polymorphisms interacting to influence human traits.

A deep learning model for predicting next-generation sequencing depth from DNA sequence

Targeted high-throughput DNA sequencing is a primary approach for genomics and molecular diagnostics, and more recently as a readout for DNA information storage. Oligonucleotide probes used to enrich gene loci of interest have different hybridization kinetics, resulting in non-uniform coverage that increases sequencing costs and decreases sequencing sensitivities. Here, we present a deep learning model (DLM) for predicting Next-Generation Sequencing (NGS) depth from DNA probe sequences. Our DLM includes a bidirectional recurrent neural network that takes as input both DNA nucleotide identities as well as the calculated probability of the nucleotide being unpaired. We apply our DLM to three different NGS panels: a 39,145-plex panel for human single nucleotide polymorphisms (SNP), a 2000-plex panel for human long non-coding RNA (lncRNA), and a 7373-plex panel targeting non-human sequences for DNA information storage. In cross-validation, our DLM predicts sequencing depth to within a factor of 3 with 93% accuracy for the SNP panel, and 99% accuracy for the non-human panel. In independent testing, the DLM predicts the lncRNA panel with 89% accuracy when trained on the SNP panel. The same model is also effective at predicting the measured single-plex kinetic rate constants of DNA hybridization and strand displacement.

Classifying single nucleotide polymorphisms in humans

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation amongst the human population and are key to personalized medicine. New tests are presented to distinguish pathogenic/malign (i.e., likely to contribute to or cause a disease) from nonpathogenic/benign SNPs, regardless of whether they occur in coding (exon) or noncoding (intron) regions in the human genome. The tests are based on the nearest neighbor (NN) model of Gibbs freeenergy landscapes of DNA hybridization and on deep structural properties of DNA revealed by an approximating metric (the h-distance) in DNA spaces of oligonucleotides of a common size. The quality assessments show that the newly defined PNPG test can classify a SNP with an accuracy about 73% for the required parameters. The best performance among machine learning models is a feed-forward neural network with fivefold cross-validation accuracy of at least 73%. These results may provide valuable tools to solve the SNP classification problem, where tools are lacking, to assess the likelihood of disease causing in unclassified SNPs. These tests highlight the significance of hybridization chemistry in SNPs. They can be applied to further the effectiveness of research in the areas of genomics and metabolomics.

The Intergenerational Transmission of Developmental Nicotine Exposure-Induced Neurodevelopmental Disorder-Like Phenotypes is Modulated by the Chrna5 D397N Polymorphism in Adolescent Mice.

Maternal tobacco smoking during pregnancy constitutes developmental nicotine exposure (DNE) and is associated with nicotine dependence and neurodevelopmental disorders in both children and grandchildren as well as animal models thereof. Genetic variants such as the CHRNA5 single nucleotide polymorphism (SNP) rs16969968, which leads to an aspartic acid to asparagine substitution at amino acid position 398 (D398N) in the alpha-5 nicotinic acetylcholine receptor subunit, can also confer risk for nicotine dependence and neurodevelopmental disorders in the absence of DNE. However, the degrees to which, the consequences of maternal smoking on offspring outcomes are influenced by genetic variants and interactions therewith are not well understood. Addressing this void in the literature, the present study utilizes a DNE mouse model engineered to possess the equivalent of the human D398N SNP in CHRNA5 (D397N SNP in mice) to assess how the N397 risk allele impacts the induction and intergenerational transmission of a range of neurodevelopmental disorder-related behavioral phenotypes in first- and second-generation DNE offspring. Results reveal that offspring possessing the N397 variant in the absence of DNE as well as DNE offspring and grand offspring possessing theD397 variant exhibit analogous neurodevelopmental disorder-like phenotypes including hyperactivity, risk-taking behaviors, aberrant rhythmicity of activity, and enhanced nicotine consumption. DNE amplified these behavioral anomalies in first-generation N397 progeny, but the severity of DNE-evoked behavioral perturbations did not significantly differ between first-generation D397 and N397 DNE mice for any measure. Remarkably, the behavioral profiles of second-generation N397 DNE progeny closely resembled DNE-naive D397 mice, suggesting that the N397 variant may protect against the intergenerational transmission of DNE-induced neurodevelopmental disorder-like behaviors.

Association between intelectin-1 variation and human kidney stone disease in northeastern Thai population.

An interplay of multiple genetic and environmental factors implicates an incidence of human kidney stone disease (KSD). However, the genetic factors associated with KSD are not completely known or understood. To identify KSD-associated genetic variations among the northeastern Thai patients, a genome-wide association study (GWAS) was conducted. We initially employed genotyping of single nucleotide polymorphism (SNP) using Genome-Wide Human SNP Array 6.0 in 105 patients and in 105 normal control subjects. To overcome the limitation of small sample size, we set forth to analyze SNPs as clusters based on the concept of linkage disequilibrium (LD) and haplotype. Using this analysis, 29 genes were identified. Three candidate SNPs, including rs2039415, rs2274907, and rs3747515, were selected on the basis of haplotype analysis, potentially functional SNPs, and the functions of associated genes. Further genotyping of these SNPs in a larger sample size (altogether 216 patients and 216 control subjects) showed that the candidate SNP rs2274907 remained significantly different between case and control subjects in both genotype frequencies (OR 2.44, 95% CI 1.38-4.30; p = 0.0015) and allele frequencies (OR 1.54, 95% CI 1.17-2.03; p = 0.0021). The non-synonymous SNP rs2274907 (c.326T > A) located in exon 4 of the ITLN1 gene results in a substitution of valine (V) by aspartate (D) at position 109 (p.V109D). This substitution could affect the predicted hydrogen (H)-bonds between lysine (K) 107 and glutamine (Q) 104, which supports its association with KSD in this population.

Human Immunotypes Impose Selection on Viral Genotypes Through Viral Epitope Specificity.

Understanding the genetic interplay between human hosts and infectious pathogens is crucial for how we interpret virulence factors. Here, we tested for associations between HIV and host genetics, and interactive genetic effects on viral load (VL) in HIV-positive antiretroviral treatment-naive clinical trial participants. HIV genomes were sequenced and the encoded amino acid (AA) variants were associated with VL, human single nucleotide polymorphisms (SNPs), and imputed HLA alleles using generalized linear models with Bonferroni correction. Human (388 501 SNPs) and HIV (3010 variants) genetic data were available for 2122 persons. Four HIV variants were associated with VL (P < 1.66 × 10-5). Twelve HIV variants were associated with a range of 1-512 human SNPs (P < 4.28 × 10-11). We found 46 associations between HLA alleles and HIV variants (P < 1.29 × 10-7). HIV variants and immunotypes when analyzed separately were associated with lower VL, whereas the opposite was true when analyzed in concert. Epitope binding predictions supported our observations. Our results show the importance of immunotype specificity on viral antigenic determinants, and the identified genetic interplay emphasizes that viral and human genetics should be studied in the context of each other.Clinical Trials Registration: NCT00867048.

A cell culture model/mimic to investigate the biochemical consequences of cdkal1 mutations that result in unprocessed, non‐functional insulin and Type 2 Diabetes Mellitus

The primary objective of the project is to develop a cell culture model that mimics the pancreatic beta islets to study the biochemical consequences of single nucleotide polymorphisms (SNPs) found in the intron-5 of cdkal1 gene which codes for a tRNA modification enzyme. SNPs in cdkal1 gene has been pointed out for its role in the development of Type 2 Diabetes Mellitus (T2DM) from past few years by human Genome Wide Association Studies. Still we lack a model system to investigate the consequences of cdkal1 mutations that result in unprocessed, non-functional insulin leading to the development of T2DM. Hypothesis Incorrectly processed cdkal1 mRNA results in untranslated or incorrectly translated Cdkal1 protein. Cdkal1 protein is a thiomethyl transferase that adds an ms2 moiety to the already modified N6-threonyl carbamoyl adenosine (t6A37) in the 37 position of tRNA. Improperly processed Cdkal1 protein will not be able to modify tRNALys3. Proinsulin will not be correctly translated and processed resulting in little or no insulin secreted from the β- cells in pancreatic islets. To uncover the pathogenesis of the human cdkal1-SNP associated T2DM, the current project will produce a cell culture model of the cdkal1 gene SNP-associated human T2DM. With the model, we will be able to discover the biochemical properties of the insulin, if translated at all. In a cdkal1-knockout (KO) cells, we will rescue the insulin production with WT human cdkal1, and then discover the effects of intron-5 SNPs of the cdkal1 gene. SNPs could affect mRNA processing. Methods and preliminary results We have successfully knocked down the cdkal1 gene in a murine β-cell line (NIT1) inducible for insulin. Our recent knockdown studies in NIT1 cell lines showed that under KCl insulin induction, unprocessed insulin mRNA increases by ~40% relative to mature insulin mRNA (Fig. B) and cdkal1 mRNA is reduced by ~40%. Subsequent reduction in tRNA ms2 modification and insulin production was also observed upon cdkal1 knockdown. As 100% gene knockout is required to study the effects of SNPs, we now have a murine β-cell line (MIN-cdkal1-null); the parent of that cell line (MIN-cdkal1-het) produces insulin assessed with a glucose- stimulated insulin release (GSIR) assay. The MIN-cdkal1-null having a cdkal1 knockout will be an advantageous tool to evaluate the state of insulin production when Cdkal1 is not modifying tRNALys3. A variant of mini-gene reporter constructs carrying potential SNPs have been designed and will be expressed and analyzed in knockout cell lines to evaluate cdkal1 mRNA processing and splicing events. We are positioned to answer important questions that connect Cdkal1 protein biochemistry to T2DM like, are the human intronic SNPs of the cdkal1 gene cause incorrect processing of cdkal1 mRNA? What is the status of the insulin produced, if at all translated - is proinsulin unprocessed, aggregated or degraded and not secreted? In the long-term, the results of this study will stimulate new insights into the human health implications of tRNA modification enzymes and the role(s) of modifications not just in the pathogenesis of cdkal1 SNP-associated T2DM, but also in a variety of human diseases.