Functional Single Nucleotide Variants Research Articles

Base editor-mediated large-scale screening of functional mutations in bacteria for industrial phenotypes.

Base editing, the targeted introduction of point mutations into cellular DNA, holds promise for improving genome-scale functional genome screening to single-nucleotide resolution. Current efforts in prokaryotes, however, remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library, which falls far short of fully releasing the potential of base editors. Here, we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli. We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E. coli, and screened for adaptive mutations under isobutanol and furfural selective conditions. Guided by the screening results, we successfully identified several known and novel functional mutations. Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.

Association of single nucleotide variants in VEGFA and KDR with the risk and angiogenic features of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype and dependent on angiogenesis (AG), whose main effectors are VEGFA and VEGFR2. Functional single nucleotide variants (SNVs) are described in VEGFA and KDR genes. However, it still unknown whether VEGFA − 2578C/A, −2489C/T, −1154G/A, −634G/C, −460C/T and KDR-604T/C, −271G/A, +1192G/A and +1719A/T SNVs act on DLBCL risk and angiogenic features. Genomic DNA from 168 DLBCL patients and 205 controls was used for SNV genotyping. Angiogenesis was immunohistochemically assessed in tumor biopsies, with reactions for VEGFA, VEGFR2, and CD34. VEGFA –1154GG genotype were associated with 1.6-fold higher DLBCL risk. KDR + 1192GG plus KDR + 1719 TT and KDR + 1192GG plus VEGFA − 2578CC combined genotypes are associated with 2.19- and 2.04-fold higher risks of DLBCL, respectively. VEGFA − 634GG or GC genotypes are associated with increased microvessel density and VEGFA levels. No relationship was observed between SNVs and cell-of-origin classification of DLBCL, but higher VEGFA and VEGFR2 were seen in non-germinal center tumors.

Pharmacogenomics of in vitro response of the NCI-60 cancer cell line panel to Indian natural products

BackgroundIndian natural products have been anecdotally used for cancer treatment but with limited efficacy. To better understand their mechanism, we examined the publicly available data for the activity of Indian natural products in the NCI-60 cell line panel.MethodsWe examined associations of molecular genomic features in the well-characterized NCI-60 cancer cell line panel with in vitro response to treatment with 75 compounds derived from Indian plant-based natural products. We analyzed expression measures for annotated transcripts, lncRNAs, and miRNAs, and protein-changing single nucleotide variants in cancer-related genes. We also examined the similarities between cancer cell line response to Indian natural products and response to reference anti-tumor compounds recorded in a U.S. National Cancer Institute (NCI) Developmental Therapeutics Program database.ResultsHierarchical clustering based on cell line response measures identified clustering of Phyllanthus and cucurbitacin products with known anti-tumor agents with anti-mitotic mechanisms of action. Curcumin and curcuminoids mostly clustered together. We found associations of response to Indian natural products with expression of multiple genes, notably including SLC7A11 involved in solute transport and ATAD3A and ATAD3B encoding mitochondrial ATPase proteins, as well as significant associations with functional single nucleotide variants, including BRAF V600E.ConclusionThese findings suggest potential mechanisms of action and novel associations of in vitro response with gene expression and some cancer-related mutations that increase our understanding of these Indian natural products.

A Regional Burden of Sequence-Level Variation in the 22q11.2 Region Influences Schizophrenia Risk and Educational Attainment

BackgroundGenomic loci where recurrent pathogenic copy number variants are associated with psychiatric phenotypes in the population may also be sensitive to the collective impact of multiple functional low-frequency single nucleotide variants (SNVs). MethodsWe examined the cumulative impact of low-frequency, functional SNVs within the 22q11.2 region on schizophrenia risk in a discovery cohort and an independent replication cohort (N = 1933 and N = 11,128, respectively), as well as the impact on educational attainment (EA) in a third, independent, general population cohort (N = 2081). In the discovery and EA cohorts, SNVs were identified using genotyping arrays; in the replication cohort, whole-exome sequencing was available. For verification, we compared the regional SNV count for schizophrenia cases in the discovery cohort with a normative count distribution derived from a large population dataset (N = 26,500) using bootstrap procedures. ResultsIn both schizophrenia cohorts, an increased regional SNV burden (≥4 low-frequency SNVs) in the 22q11.2 region was associated with schizophrenia (discovery cohort: odds ratio = 7.48, p = .039; replication cohort: odds ratio = 1.92, p = .004). In the EA cohort, an increased regional SNV burden at 22q11.2 was associated with decreased EA (odds ratio = 4.65, p = .049). Comparing the SNV count for schizophrenia cases with a normative distribution confirmed the unique nature of the distribution for schizophrenia cases (p = .002). ConclusionsIn the general population, an increased burden of low-frequency, functional SNVs in the 22q11.2 region is associated with schizophrenia risk and a decrease in EA. These findings suggest that in addition to structural variation, a cumulative regional burden of low-frequency, functional SNVs in the 22q11.2 region can also have a relevant phenotypic impact.

Association Analysis Between the Functional Single Nucleotide Variants in miR-146a, miR-196a-2, miR-499a, and miR-612 With Acute Lymphoblastic Leukemia.

BackgroundAcute lymphoblastic leukemia (ALL) is characterized by an abnormal proliferation of immature lymphocytes, in whose development involves both environmental and genetic factors. It is well known that single nucleotide polymorphisms (SNPs) in coding and noncoding genes contribute to the susceptibility to ALL. This study aims to determine whether SNPs in miR-146a, miR-196a-2, miR-499a, and miR-612 genes are associated with the risk to ALL in pediatric Mexican population.MethodsA multicenter case-control study was carried out including patients with de novo diagnosis of ALL and healthy subjects as control group. The DNA samples were obtained from saliva and peripheral blood, and the genotyping of rs2910164, rs12803915, rs11614913, and rs3746444 was performed using the 5′exonuclease technique. Gene-gene interaction was evaluated by the multifactor dimensionality reduction (MDR) software.Results miR-499a rs3746444 showed significant differences among cases and controls. The rs3746444G allele was found as a risk factor to ALL (OR, 1.6 [95% CI, 1.05–2.5]; p = 0.028). The homozygous GG genotype of rs3746444 confers higher risk to ALL than the AA genotype (OR, 5.3 [95% CI, 1.23–23.4]; p = 0.01). Moreover, GG genotype highly increases the risk to ALL in male group (OR, 17.6 [95% CI, 1.04–298.9]; p = 0.00393). In addition, an association in a gender-dependent manner among SNPs located in miR-146a and miR-196a-2 genes and ALL susceptibility was found.ConclusionOur findings suggest that SNP located in miR-499a, miR-146a, and miR-196a-2 genes confer risk to ALL in Mexican children. Experimental analysis to decipher the role of these SNPs in human hematopoiesis could improve our understanding of the molecular mechanism underlying the development of ALL.

A14343 Whole-Exome Sequencing Identifies a coding variant in SULT1A2 Gene Associated with Obesity in Chinese Children

Objectives: Despite numerous genetic variants associated with obesity-related traits have been identified, much of the obesity heritability remains unexplained. We aimed to identify novel coding variants and genes associated with childhood obesity in China. Methods: The study comprised three stages. We conducted deep-depth (50 × ) whole exome sequencing in 18 severely obese and 15 lean children. After several filtering steps, we retained promising single nucleotide variants (SNVs) nominal association (P ≤ 0.05) with obesity, dyslipidemia or impaired plasma glucose for validation in 2,403 obese and 3,778 non-overweight children. Variants showing potential associations were confirmed by Sanger sequencing. Results: Exome sequencing identified 43,264 functional SNVs. We identified two coding variants were associated with common or central obesity at exome-wide significance: rs1059491 in the SULT1A2 gene for common obesity (P = 2.18 × 10–24, odds ratio = 2.16) and rs768847893 in the MXRA5 gene for central obesity (P = 2.57 × 10–8, odds ratio = 2.96, females only). Conclusion: Our findings demonstrate that rs1059491 is strongly associated with common obesity in Chinese children. Additionally, rs768847893 is associated with central obesity in female. Our results provide evidence of the existence of coding variants associated with obesity and have important implications for the pathophysiology of obesity.

World-wide distributions of lactase persistence alleles and the complex effects of recombination and selection

The genetic trait of lactase persistence (LP) is associated with at least five independent functional single nucleotide variants in a regulatory region about 14 kb upstream of the lactase gene [−13910*T (rs4988235), −13907*G (rs41525747), −13915*G (rs41380347), −14009*G (rs869051967) and −14010*C (rs145946881)]. These alleles have been inferred to have spread recently and present-day frequencies have been attributed to positive selection for the ability of adult humans to digest lactose without risk of symptoms of lactose intolerance. One of the inferential approaches used to estimate the level of past selection has been to determine the extent of haplotype homozygosity (EHH) of the sequence surrounding the SNP of interest. We report here new data on the frequencies of the known LP alleles in the ‘Old World’ and their haplotype lineages. We examine and confirm EHH of each of the LP alleles in relation to their distinct lineages, but also show marked EHH for one of the older haplotypes that does not carry any of the five LP alleles. The region of EHH of this (B) haplotype exactly coincides with a region of suppressed recombination that is detectable in families as well as in population data, and the results show how such suppression may have exaggerated haplotype-based measures of past selection.

Proteogenomic analysis prioritises functional single nucleotide variants in cancer samples.

Massively parallel DNA sequencing enables the detection of thousands of germline and somatic single nucleotide variants (SNVs) in cancer samples. The functional analysis of these mutations is often carried out through in silico predictions, with further downstream experimental validation rarely performed. Here, we examine the potential of using mass spectrometry-based proteomics data to further annotate the function of SNVs in cancer samples. RNA-seq and whole genome sequencing (WGS) data from Jurkat cells were used to construct a custom database of single amino acid variant (SAAV) containing peptides and identified over 1,000 such peptides in two Jurkat proteomics datasets. The analysis enabled the detection of a truncated form of splicing regulator YTHDC1 at the protein level. To extend the functional annotation further, a Jurkat phosphoproteomics dataset was analysed, identifying 463 SAAV containing phosphopeptides. Of these phosphopeptides, 24 SAAVs were found to directly impact the phosphorylation event through the creation of either a phosphorylation site or a kinase recognition motif. We identified a novel phosphorylation site created by a SAAV in splicing factor SF3B1, a protein that is frequently mutated in leukaemia. To our knowledge, this is the first study to use phosphoproteomics data to directly identify novel phosphorylation events arising from the creation of phosphorylation sites by SAAVs. Our study reveals multiple functional mutations impacting the splicing pathway in Jurkat cells and demonstrates potential benefits of an integrative proteogenomics analysis for high-throughput functional annotation of SNVs in cancer.

Genetic epidemiology of pharmacogenetic variants in South East Asian Malays using whole-genome sequences.

Expanding the scope of pharmacogenomic research by including multiple global populations is integral to building robust evidence for its clinical translation. Deep whole-genome sequencing of diverse ethnic populations provides a unique opportunity to study rare and common pharmacogenomic markers that often vary in frequency across populations. In this study, we aim to build a diverse map of pharmacogenetic variants in South East Asian (SEA) Malay population using deep whole-genome sequences of 100 healthy SEA Malay individuals. We investigated the allelic diversity of potentially deleterious pharmacogenomic variants in SEA Malay population. Our analysis revealed 227 common and 466 rare potentially functional single nucleotide variants (SNVs) in 437 pharmacogenomic genes involved in drug metabolism, transport and target genes, including 74 novel variants. This study has created one of the most comprehensive maps of pharmacogenetic markers in any population from whole genomes and will hugely benefit pharmacogenomic investigations and drug dosage recommendations in SEA Malays.

Abstract 2764: Cosegregating variants in chronic lymphocytic leukemia (CLL) families that are located in loci discovered by genome wide association studies (GWAS)

Abstract Introduction Chronic lymphocytic leukemia (CLL) is a B-cell malignancy that is known to have a familial component to disease risk. Although 31 loci have been found to be associated with CLL risk, the functional variant(s) driving these associations is mostly unknown. Here we set out to identify rare, highly-penetrant, cosegregating, susceptibility variants within the known GWAS discovered loci using whole exome sequencing (WES) data in CLL families from the Mayo Clinic family study of B-cell malignancies. Methods We performed WES on germ line DNA of 93 CLL families with two or more members with CLL, using Agilent capture kits and Illumina HiSeq2000. Bioinformatics analyses leveraged the following software packages: Novoalign, Picard, The Genome Analysis Toolkit (GATK), and the Biological Reference Repository (bioR). Quality control filters were implemented; subjects with mis-specified relationships were removed, as were variants with <75% call rate, <8X coverage, and those identified as sequencing artifacts. Each GWAS locus was defined by +/- 1Mb of the top GWAS hit within the locus. Linkage disequilibrium (LD) was calculated among the single nucleotide variants (SNVs) located within each locus. Potentially functional SNVs were identified based on: a) uncommon in public databases (< 5%), b) cosegregating in at least two CLL families, c) being highly conserved and in coding regions, and d) functional prediction status of deleterious (SIFT Score), damaging (PolyPhen Score), and a moderate, or high variant impact (SNP Effect). Results In our 93 CLL families, we sequenced 443 individuals: 160 with CLL, 73 with monoclonal B cell lymphocytosis (MBL), and 210 relatives that were not diagnosed with CLL or MBL at the time of sequencing. Median age of CLL diagnosis was 59 years (range 34-87), and 56% were male. Among the MBL individuals and relatives, the median age at recruitment was 55 years (range 18-93), and 40% were male. A total of 317,666 SNVs passed our sequencing quality control filters of which 10,731 were within +/- 1 Mb of known GWAS hits from 31 loci. Of these SNVs, 91% were in coding regions, 18% were reported to have high or moderate impact, 6% were estimated to be damaging and 6% were predicted to be deleterious. From these SNVs, we identified 76 putatively functional variants distributed across 25 GWAS loci that were cosegregating in the individuals with CLL or MBL in multiple CLL pedigrees. These SNVs were all located in coding regions with high or moderate impact and were predicted to be damaging and deleterious. Of these 76 variants, 56 had a frequency of <0.005 in 1000 Genomes’ European population while the remaining 20 had a frequency of 1%. Conclusions Through WES, we identified a number of rare, penetrant and potentially predisposing SNVs located within 25 of the 31 CLL GWAS-discovered loci. These segregating variants provide a list for future validation and functional studies. Citation Format: Sara Beiggi, Daniel R. O'Brien, Sara J. Achenbach, Kari G. Chaffee, Timothy G. Call, Neil E. Kay, Tait D. Shanafelt, Julie Cunningham, James R. Cerhan, Celine M. Vachon, Susan L. Slager. Cosegregating variants in chronic lymphocytic leukemia (CLL) families that are located in loci discovered by genome wide association studies (GWAS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2764. doi:10.1158/1538-7445.AM2015-2764

Exome Sequencing in Chronic Lymphocytic Leukemia (CLL) and Multiple Myeloma (MM) Families Identifies Cosegregating Functional Variants

Introduction: Several B-cell malignancies, including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are known to have a familial component to disease risk. Although several common inherited genetic variants have been found to be associated with risk of these malignancies, a large proportion of the heritability remains unexplained. Here we set out to identify rare high-penetrance susceptibility variants for CLL and MM using the established Mayo Clinic family study of B-cell malignancies.Methods: We performed exome sequencing on germ line DNA of 106 families with either two or more members with CLL (n=93 families) or two or more members with MM or monoclonal gammopathy of undetermined significance (MGUS) (n=13 families) using Aglient capture kits. All samples were sequenced on an Illumina HiSeq2000. Bioinformatics analyses leveraged the following software packages: Novoalign, Picard, The Genome Analysis Toolkit (GATK), and the Biological Reference Repository (bioR). Quality control filters were implemented; subjects with mis-specified relationships were removed as were variants with <95% call rate or <20x coverage, and those variants identified as sequencing artifacts. To identify potentially functional single nucleotide variants, the following was required a) uncommon in public databases, b) cosegregating variants in multiple families, c) variants to be highly conserved and in coding regions, and d) functional prediction status of deleterious (SIFT score), damaging (PolyPhen score), and a moderate, or high variant impact (SNPEffect). The association of these variants with risk was then evaluated in large samples of cases and controls in genome-wide association studies (GWAS).Results: In the CLL familial cohort, there were 443 individuals (160 with CLL, 73 with monoclonal B cell lymphocytosis (MBL), and 210 relatives without CLL or MBL) sequenced; and in the MM cohort, there were 39 individuals (13 with MM, 13 with MGUS and 13 without MM or MGUS) sequenced. A total of 61,992 single nucleotide variants passed quality control filters in the CLL cohort and 39,456 single nucleotide variants in the MM cohort. From these, we identified 32 variants in the CLL cohort and 39 variants in the MM cohort that were cosegregating in multiple CLL or MM families, respectively, and were predicted to affect protein structure or function. Of these, one CLL variant was within 1 Mb of a known GWAS-discovered locus. Moreover, there were seven CLL and four MM variants that were within 1 Mb of single nucleotide polymorphisms (SNPs) that had statistical significance between p<10-4 and p<10-6 in GWAS. Finally, there were 3 variants found to be common between CLL and MM families (see Table).Conclusions: Through whole exome sequencing, we identified a number of rare, highly penetrant predisposition variants for CLL and MM. Several of these variants were common to both B-cell malignancies providing evidence of shared genetic components; one variant may be a functional driver of the GWAS-discovered loci. These identified variants provide a list for future validation studies.Table:Exome variants common in both Chronic Lymphocytic Leukemia and Multiple MyelomaSNP IDrs41314099rs61754114rs143025033Chromosome1767GeneWSCD1BACH2TNS3Penetrance (CLL)0.700.750.80Penetrance (MM)1.000.800.75Allele Frequency (CLL)0.0150.0250.017Allele Frequency (MM)0.0770.0770.0581000 Genome Frequency (European)0.0100.0200.010SIFT TermDeleteriousDeleteriousDeleteriousPolyPhen TermProbably DamagingProbably DamagingProbably Damaging SNPSingle Nucleotide Polymorphism; CLL: Chronic Lymphocytic Leukemia; MM: Multiple Myeloma; Penetrance is defined as proportion of individuals carrying the alternate allele that are also affected; SIFT: Sorting Intolerant From Tolerant, predicts whether an amino acid substitution affects protein function; PolyPhen: Polymorphism Phenotyping, predicts possible impact of an amino acid substitution on the structure of a human protein DisclosuresNo relevant conflicts of interest to declare.

Identifying bipolar disorder susceptibility loci in a densely affected pedigree

Identifying bipolar disorder susceptibility loci in a densely affected pedigree

Extended Evaluation of Serotonin Transporter Gene Functional Polymorphisms in Subjects with Post-Stroke Depression

As an extension of our previous observation, relating a serotonin transporter gene-linked promoter region (5-HTTLPR) diallelic functional polymorphism (short [S] and long [L] alleles) to the risk of post-stroke major depression (PSD), this study investigated the role of 2 other functional polymorphisms of the serotonin transporter gene (5-HTT) in the same sample of subjects with PSD. In a clinical sample of 26 patients with PSD and 25 unrelated nondepressed stroke patients of Caucasian descent, we examined the frequencies of a functional single nucleotide variant (A/G) within the promoter region (rs25531) and located in L (16-repeat) and S (14-repeat) alleles of 5-HTTLPR, and a variable number tandem repeat (VNTR) polymorphism in intron 2. There were significant intergroup differences in the allelic frequencies of 5-HTTLPR/rs25531 (SA, LA, and LG) (P < 0.05) and in the combined frequencies of lower-expressing alleles (SA and LG) and higher-expressing alleles (LA) (P < 0.025) between subjects with PSD and nondepressed stroke. However, the differences in the combined frequencies of lower-expressing (SA/SA, SA/LG, and LG/LG), intermediate-expressing (SA/LA and LA/LG), and higher-expressing (LA/LA) genotypes of 5-HTTLPR were not significant. Further, no significant intergroup differences were found in the allelic and genotypic frequencies of the intron 2 VNTR. These findings strengthen the support for an association between PSD and lower-expressing alleles of 5-HTTLPR.

Novel 5-HTTLPR Allele Associates with Higher Serotonin Transporter Binding in Putamen: A [ 11C] DASB Positron Emission Tomography Study

The serotonin transporter (5-HTT)-linked polymorphic region (5-HTTLPR) has two frequent alleles, designated long (L), and short (S). The S allele is associated with lower levels of 5-HTT mRNA and lower 5-HTT expression in human cell lines. A functional single nucleotide variant was detected within L, designated L(A) and L(G). Only L(A) is associated with high levels of in vitro 5-HTT expression, whereas L(G) is low expressing and more similar to S. We examined the possible influence of the long (A/G) variant on 5-HTT density in the living human brain using 3-(11)C-amino-4-(2-dimethylaminomethylphenyl-sulfanyl) benzonitrile ([(11)C]DASB) positron emission tomography. The 5-HTT binding potential (5-HTT BP), an index of 5-HTT density, was found in 43 healthy subjects genotyped for 5-HTTLPR long (A/G), and in an ethnically homogenous subsample of 30 Caucasian-Canadians. The L(A)/L(A) was associated with higher 5-HTT BP in putamen (p = .026, not corrected). This association became stronger in the Caucasian subsample (p = .004) and was significant even after correcting for multiple comparisons. The 5-HTTLPR long (A/G) polymorphism influences 5-HTT density leading to higher putamen 5-HTT BP in healthy L(A)/L(A) carriers of Caucasian ancestry. This finding extends the role of this polymorphism from in vitro reports of higher 5-HTT expression with the L(A)/L(A) genotype into in vivo brains of healthy human subjects.