Allele-specific Assay Research Articles

Donor Genotype in the Interleukin-7 Receptor α-Chain Predicts Risk of Graft-versus-Host Disease and Cytomegalovirus Infection after Allogeneic Hematopoietic Stem Cell Transplantation.

The efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) is challenged by acute and chronic graft-versus-host disease (aGVHD and cGVHD) and viral infections due to long-lasting immunodeficiency. Interleukin-7 (IL-7) is a cytokine essential for de novo T cell generation in thymus and peripheral T cell homeostasis. In this study, we investigated the impact of the single nucleotide polymorphism rs6897932 in the IL-7 receptor α-chain (IL-7Rα) which has previously been associated with several autoimmune diseases. We included 460 patients undergoing allogeneic HSCT after a myeloablative conditioning. Patients had a median age of 26.3 years (0.3–67.0 years), and 372 (80.9%) underwent HSCT for malignant diseases. Donors were matched sibling donors (n = 147), matched unrelated donors (n = 244) or mismatched unrelated donors (n = 69), and the stem cell source were either bone marrow (n = 329) or peripheral blood (n = 131). DNA from donors was genotyped for the IL-7Rα single nucleotide polymorphism (SNP) rs6897932 using an allele-specific primer extension assay (CC: n = 252, CT: n = 178, TT: n = 30). The donor T allele was associated with a higher risk of grades III–IV aGVHD (HR = 2.0, 95% CI = 1.1–3.8, P = 0.034) and with significantly increased risk of extensive cGVHD (HR = 2.0, 95% CI = 1.1–3.6, P = 0.025) after adjustment for potential risk factors. In addition, the TT genotype was associated with a higher risk of cytomegalovirus (CMV) infection post-transplant (HR = 2.4, 95% CI = 1.2–4.3, P = 0.0068). Numbers of T cells were significantly higher on day +60 in patients receiving a rs6897932 TT graft (CD3+: 109% increase, P = 0.0096; CD4+: 64% increase, P = 0.038; CD8+: 133% increase, P = 0.011). Donor heterozygosity for the T allele was associated with inferior overall survival (HR = 1.7, 95% CI = 1.2–2.3, P = 0.0027) and increased treatment-related mortality (HR = 2.3, 95% CI = 1.3–4.0, P = 0.0047), but was not associated with the risk of relapse (P = 0.35). In conclusion, the IL-7Rα rs6897932 genotype of the donor is predictive of aGVHD and cGVHD, CMV infection, and mortality following HSCT. These findings indicate that IL-7Rα SNP typing of donors may optimize donor selection and facilitate individualization of treatment in order to limit treatment-related complications.

A Resource for the Allele-Specific Analysis of DNA Methylation at Multiple Genomically Imprinted Loci in Mice.

Genomically imprinted loci are expressed mono-allelically, dependent upon the parent of origin. Their regulation not only illuminates how chromatin regulates gene expression but also how chromatin can be reprogrammed every generation. Because of their distinct parent-of-origin regulation, analysis of imprinted loci can be difficult. Single nucleotide polymorphisms (SNPs) are required to accurately assess these elements allele specifically. However, publicly available SNP databases lack robust verification, making analysis of imprinting difficult. In addition, the allele-specific imprinting assays that have been developed employ different mouse strains, making it difficult to systemically analyze these loci. Here, we have generated a resource that will allow the allele-specific analysis of many significant imprinted loci in a single hybrid strain of Mus musculus. This resource includes verification of SNPs present within 10 of the most widely used imprinting control regions and allele-specific DNA methylation assays for each gene in a C57BL/6J and CAST/EiJ hybrid strain background.

Abstract LB-B03: Exosome-based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients

Abstract About 60% of non-small cell lung cancer (NSCLC) patients develop resistance to targeted epidermal growth factor receptor (EGFR) inhibitor therapy through the EGFR T790M mutation. Although these patients respond well to third generation tyrosine kinase inhibitors like osimertinib, obtaining tissue biopsies for mutation profiling poses risks and is often not feasible. Detecting the mutation in a liquid biopsy from plasma has emerged as a viable alternative to biopsy, however existing liquid biopsy tests using cell-free DNA (cfDNA) often suffer from low sensitivity due to the low copy number of mutant molecules in circulation. As an example, the Food and Drug Adminstration (FDA) approved companion diagnostic for T790M mutation status in plasma (cobas® EGFR mutation Test version 2) only has a 58% sensitivity at a specificity of 80%. In this study we aimed to achieve a higher performance by combining the mutated alleles on cfDNA as well as exosomal RNA and DNA (exoNA) . ExoNA and cfDNA were extracted from the plasma of NSCLC patients with biopsy-confirmed T790M-positive (N = 102) or T790M-negative (N = 108) samples. The T790M mutation status in plasma exoNA and cfDNA was determined using an analytically validated allele-specific qPCR assay in a CLIA laboratory. The training cohort consisted of 105 samples (51 positives, 54 negatives). The validation cohort consisted of 105 samples (51 positives, 54 negatives), where ~40% of the patients had intrathoracic disease(M0/M1a). Detection of the T790M mutation in exoNA and cfDNA achieved 92% sensitivity and 89% specificity when compared to tumor biopsy results in the validation cohort. We also observed a very high sensitivity (88%) in patients with intrathoracic disease (M0/M1a), patients for whom detection by liquid biopsy has been proven particularly challenging. The combination of exoNA and cfDNA for EGFR T790M detection has a higher sensitivity and specificity compared to historical cohorts using ctDNA alone. This is especially important as it could aid giving the right treatment to patients where biopsy is not available or avoid unnecessary tumor biopsies for T790M mutation testing. Citation Format: Elena Castellanos, Dominik G. Grimm, Vasisht Tadigotla, Stefan Bentink, James Hurley, John Healy, Patricia L. Neal, Mia Sher, Dan Baughman, Chris Karlovich, Mitch Raponi, Anne Krug, Mikkel Noerholm, Luis E. Raez, Johan Skog. Exosome-based Detection of EGFR T790M in Plasma from Non-Small Cell Lung Cancer Patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B03.

Genotyping by Sequencing in Almond: SNP Discovery, Linkage Mapping, and Marker Design.

In crop plant genetics, linkage maps provide the basis for the mapping of loci that affect important traits and for the selection of markers to be applied in crop improvement. In outcrossing species such as almond (Prunus dulcis Mill. D. A. Webb), application of a double pseudotestcross mapping approach to the F1 progeny of a biparental cross leads to the construction of a linkage map for each parent. Here, we report on the application of genotyping by sequencing to discover and map single nucleotide polymorphisms in the almond cultivars “Nonpareil” and “Lauranne.” Allele-specific marker assays were developed for 309 tag pairs. Application of these assays to 231 Nonpareil × Lauranne F1 progeny provided robust linkage maps for each parent. Analysis of phenotypic data for shell hardness demonstrated the utility of these maps for quantitative trait locus mapping. Comparison of these maps to the peach genome assembly confirmed high synteny and collinearity between the peach and almond genomes. The marker assays were applied to progeny from several other Nonpareil crosses, providing the basis for a composite linkage map of Nonpareil. Applications of the assays to a panel of almond clones and a panel of rootstocks used for almond production demonstrated the broad applicability of the markers and provide subsets of markers that could be used to discriminate among accessions. The sequence-based linkage maps and single nucleotide polymorphism assays presented here could be useful resources for the genetic analysis and genetic improvement of almond.

P3.01-041 The Robustness of Allele-Specific qPCR Assays for Detection of EGFR Mutations in Plasma Cell-Free DNA from NSCLC Patients

P3.01-041 The Robustness of Allele-Specific qPCR Assays for Detection of EGFR Mutations in Plasma Cell-Free DNA from NSCLC Patients

Heterogeneity of BRAF, NRAS, and TERT Promoter Mutational Status in Multiple Melanomas and Association with MC1R Genotype: Findings from Molecular and Immunohistochemical Analysis

Data on somatic heterogeneity and germline-somatic interaction in multiple primary melanoma (MPM) patients are limited. We investigated the mutational status of BRAF, NRAS, and TERT promoter genes in 97 melanomas of 44 MPM patients and compared molecular and immunohistochemical findings. We further evaluated the association of somatic alterations with the germline MC1R genotype. Mutations in BRAF gene were identified in 41.2% (40/97) of melanomas, in NRAS in 2.1% (2/97), and in TERT promoter in 19.6% (19/97). Distribution of BRAF mutations did not differ across multiple melanomas (P=0.85), whereas TERT promoter changes decreased from first to subsequent melanomas (P=0.04). Intrapatient discrepancy of BRAF mutations among multiple tumors was detected in 14 of 44 MPM patients (32%) and of BRAF/NRAS/TERT promoter genes in 20 of 44 (45%). We observed a high rate of agreement between allele-specific TaqMan assay and immunohistochemistry in BRAFV600E detection (κ=0.83, P<0.01) with 86 of 97 melanomas (88.7%) presenting similar BRAF status. Germline MC1R variants were identified in 81.4% (35/43) of MPM patients with no association of MC1R genotype with somatic mutations or with intrapatient concordance of somatic mutational profile. Our results support the genetic diversity of multiple melanomas and show that somatic heterogeneity is not influenced by inherited MC1R variants. Immunohistochemistry may be useful as an initial screening test.

Association Between a CCL17 Genetic Variant and Risk of Coronary Artery Disease in a Chinese Han Population.

In the present study we investigated the effects of genetic variations in the C-C motif chemokine ligand 17 (CCL17) gene on serum CCL17 levels and risk of coronary artery disease (CAD).Methods and Results:A case-control study was conducted to determine causal inferences amongCCL17single-nucleotide polymorphisms (SNPs), serum CCL17 levels, and risk of CAD. Luciferase assays, electrophoretic mobility shift assays (EMSA), and allele-specific quantitative chromatin immunoprecipitation (ChIP) assays were used to assess the function of the SNPs. In all, 947 participants (794 with CAD, 153 without CAD) were included in the study. The T allele in rs223828, located in intron of theCCL17gene, was associated with increased serum CCL17 levels as well as increased CAD risk. A causal inference test using mediation analysis suggested that rs223828 had a significant indirect casual effect on the increased risk of CAD mediated via serum CCL17 levels. Luciferase assays confirmed that the rs223828T allele enhancesCCL17promoter activity. Protein-DNA binding studies using EMSA and allele-specific quantitative ChIP assays indicated preferential activator protein-1 (AP-1) complex formation and recruitment with the rs223828 T allele compared with the C allele. We propose that theCCL17SNP rs223828 is associated with increased risk of CAD, and that this site may be a potential AP-1 binding site.

Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway.

Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target BHLHE41. The alignment of multiple sites in the HIF cis-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.

AGE MODERATES EFFECTS OF PICALM RS541458 ON COGNITIVE IMPAIRMENT AND BRAIN STRUCTURE AND FUNCTIONS

The PICALMrs541458 T allele has been recognized as a risk factor for late-onset Alzheimer's disease. And age might modulate the effects of genetic factors on cognitive functions and brain. Thus, the current study intended to examine whether the effects of rs541458 polymorphism on cognitive functions, resting state functional connectivity and gray matter volume would be modulated by age in Chinese non-demented elderly. We enrolled 657 non-demented old subjects to evaluate their cognitions through a series of neuropsychological tests and to detect rs541458 and APOE genotype through their blood samples using Taqman allele-specific assays. Among them, 78 participants received T1-weighted structural imaging and resting state functional magnetic resonance imaging. The results from neuropsychological tests showed that interactive effects of rs541458×age exhibited on executive function and processing speed after controlling for gender, years of education and APOEε4 status. The effects of rs541458 on resting state functional connectivity of the left superior parietal gyrus within left frontal-parietal network and gray matter volume of left middle temporal gyrus were modulated by age as well. Furthermore, the reduction of functional connectivity of superior parietal gyrus was closely related with better executive function in the T allele carriers-<65 years old group. And the increased volume of left middle temporal gyrus was significantly related with better executive function in the two CC genotype groups.

Introduction of High Throughput and Cost Effective SNP Genotyping Platforms in Soybean

We presented here the application of two in-plate SNP (single nucleotide polymorphism) genotyping platforms for soybean plants [Glycine max (L.) Merr.], KASP® (Kompetitive Allele Specific PCR genotyping, LGC Genomics) and TaqMan® (Life Technologies) respectively. These two systems offer us an ability to determine the genotypes of 384 individual samples accurately and efficiently by allele specific PCR in a single plate using typical PCR conditions. Both of the systems require small quantity of genomic DNA obtained from a simple DNA extraction. The genomic sequences containing target SNPs can easily be used as a basic blueprint to design the probes and primers of KASP® and TaqMan® assays whether the sequences are obtained from the genome sequence of soybean William 82 (Wm82.a2.v1), Illumina Soy50k SNPs, or parallel resequencing. Moreover, we listed the pros and cons of the two systems and explained the principles behind the platforms. The high call rate and clear clustering separation of the SNPs can be readily obtained from these platforms without conducting any assay optimization processes. These platforms can routinely be performed on 96/384-well plate format with or without an automation procedure. Therefore, these platforms are especially suitable for the SNP genotyping on a particular trait with a large sample size, gene fine mapping, and marker assisted selection. Further, they require little hands-on experience and achieve per-site and per-individual costs below that of current SSR, AFLP, RFLP, and SNP chip technologies. The platforms can be used for genotyping on a wide range of organisms due to their simplicity and flexibility of handling. Meanwhile, we also especially presented some of the advantages using KASP® SNP genotyping pipeline, which was cost effective in the selection of allele specific assay and therefore, efficiently facilitated the soybean genotyping across large numbers (thousands or more) of individual lines for a great range of markers (hundreds to thousands) in our laboratory.

Abstract P6-07-20: Allele-specific PCR assay for estrogen receptor (ESR1) mutation detection

Abstract Seventy percent of breast cancers are estrogen receptor (ER) positive and, while the majority of these patients initially respond to hormone therapy, approximately 20-30% will become therapy refractory. Recent data suggest activating-mutations in the estrogen receptor gene (ESR1) ligand binding domain (LBD), which are acquired during anti-estrogen treatment and rarely found in primary untreated ER+ breast cancer, are associated with resistance. Some researchers propose that ESR1 mutations are a prognostic and predictive marker, but more studies are needed to assess the clinical utility of the ESR1 mutations. However, current ESR1 mutation detection methods, that is, next generation sequencing and digital PCR, are very labor-intensive, lengthy, and expensive and require special training. To aid research on the diagnostic potential of ESR1 mutations, we developed a prototype allele-specific qPCR assay for the detection of 18 recurrent mutations in the ESR1 LBD. The test is suitable for DNA obtained from FFPE tissue as well cfDNA from plasma. It is sensitive, yet simple, robust and results can be obtained in less than 8 hours. Herein, we demonstrate the analytical performance of our ESR1 mutation detection assay using contrived samples. We are also planning to test clinical FFPET and plasma samples in the future. Citation Format: Litterst CM, Tran HB, Chien S, Chen X, Wen W, Begovich A. Allele-specific PCR assay for estrogen receptor (ESR1) mutation detection [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-20.

Novel allele-specific quantification methods reveal no effects of adult onset CAG repeats on HTT mRNA and protein levels.

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149109767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.

Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells.

We developed an allele-specific assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to genotype and profile active regulatory DNA across the genome. Using a mouse hybrid F1 system, we found that monoallelic DNA accessibility across autosomes was pervasive, developmentally programmed and composed of several patterns. Genetically determined accessibility was enriched at distal enhancers, but random monoallelically accessible (RAMA) elements were enriched at promoters and may act as gatekeepers of monoallelic mRNA expression. Allelic choice at RAMA elements was stable across cell generations and bookmarked through mitosis. RAMA elements in neural progenitor cells were biallelically accessible in embryonic stem cells but premarked with bivalent histone modifications; one allele was silenced during differentiation. Quantitative analysis indicated that allelic choice at the majority of RAMA elements is consistent with a stochastic process; however, up to 30% of RAMA elements may deviate from the expected pattern, suggesting a regulated or counting mechanism.

Genetic diagnosis of α1-antitrypsin deficiency using DNA from buccal swab and serum samples.

α1-Antitrypsin deficiency (AATD) is associated with a high risk of developing lung and liver disease. Despite being one of the most common hereditary disorders worldwide, AATD remains under-diagnosed and prolonged delays in diagnosis are usual. The aim of this study was to validate the use of buccal swab samples and serum circulating DNA for the complete laboratory study of AATD. Sixteen buccal swab samples from previously characterized AATD patients were analyzed using an allele-specific genotyping assay and sequencing method. In addition, 19 patients were characterized by quantification, phenotyping and genotyping using only serum samples. The 16 buccal swab samples were correctly characterized by genotyping. Definitive results were obtained in the 19 serum samples analyzed by quantification, phenotyping and genotyping, thereby performing the complete AATD diagnostic algorithm. Buccal swab samples may be useful to expand AATD screening programs and family studies. Genotyping using DNA from serum samples permits the application of the complete diagnostic algorithm without delay. These two methods will be useful for obtaining more in depth knowledge of the real prevalence of patients with AATD.

Genetic Biomarker Prevalence Is Similar in Fecal Immunochemical Test Positive and Negative Colorectal Cancer Tissue

Fecal immunochemical test (FIT) screening detects most asymptomatic colorectal cancers. Combining FIT screening with stool-based genetic biomarkers increases sensitivity for cancer, but whether DNA biomarkers (biomarkers) differ for cancers detected versus missed by FIT screening has not been evaluated in a community-based population. To evaluate tissue biomarkers among Kaiser Permanente Northern California patients diagnosed with colorectal cancer within 2years after FIT screening. FIT-negative and FIT-positive colorectal cancer patients 50-77years of age were matched on age, sex, and cancer stage. Adequate DNA was isolated from paraffin-embedded specimens in 210 FIT-negative and 211 FIT-positive patients. Quantitative allele-specific real-time target and signal amplification assays were performed for 7 K-ras mutations and 10 aberrantly methylated DNA biomarkers (NDRG4, BMP3, SFMBT2_895, SFMBT2_896, SFMBT2_897, CHST2_7890, PDGFD, VAV3, DTX1, CHST2_7889). One or more biomarkers were found in 414 of 421 CRCs (98.3%). Biomarker expression was not associated with FIT status, with the exception of higher SFMBT2_897 expression in FIT-negative (194 of 210; 92.4%) than in FIT-positive cancers (180 of 211; 85.3%; p=0.02). There were no consistent differences in biomarker expression by FIT status within age, sex, stage, and cancer location subgroups. The biomarkers of a currently in-use multi-target stool DNA test (K-ras, NDRG4, and BMP3) and eight newly characterized methylated biomarkers were commonly expressed in tumor tissue specimens, independent of FIT result. Additional study using stool-based testing with these new biomarkers will allow assessment of sensitivity, specificity, and clinical utility.

Laboratory Diagnosis by Genotyping.

Alpha-1 antitrypsin (AAT) genotyping is useful to confirm the clinical diagnosis of AAT deficiency and determine the specific allelic variant. Genotyping is the reference standard procedure for identifying rare allelic variants and characterizing new variants. It is also useful when there is a discrepancy between the patients' AAT levels and their phenotypes. AAT genotype is determined by an allele-specific genotyping assay for the S, Z, and Mmalton variants and by exome sequencing.

M33 - GENETIC VARIATION AND EPIGENETIC MODIFICATION OF SKA2: ASSOCIATIONS WITH OBSESSIVE-COMPULSIVE DISORDER DISEASE RISK AND SYMPTOM SEVERITY

Background Obsessive-Compulsive Disorder (OCD) has a strong genetic component, is highly comorbid with Major Depressive Disorder (MDD), and is often triggered by environmental stress. Epigenetic modifications are thought to play a significant role in complex psychiatric disorders and may impact the relationship between genes and the environment via the hypothalamic-pituitary-adrenal (HPA) axis. The SKA2 gene interacts with the glucocorticoid receptor, is implicated in mediating HPA axis function, but has yet to be examined in OCD. We hypothesized that genetic and epigenetic variation in SKA2 may play a role in OCD disease risk and symptom severity. Methods A sample of n=64 OCD patients, divided equally by presence/absence of comorbid MDD, and age and gender matched, were selected. Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) score was used to measure disease severity. Genotyping for the SKA2 rs7208505 marker was performed using the Taqman allele specific assay method and DNA methylation levels were quantified using bisulfite pyrosequencing. Methylation percentage was corrected for genotype using previously established methods. Genotypes for rs7208505 were compared to controls of European ancestry (n=365; 1000 Genomes Project Consortium) using Pearson’s chi-squared test. The relationship among the rs7208505 variant, methylation corrected, symptom severity, and MDD status was modeled using linear regression. Results The SKA2 rs7208505 genotype distributions significantly differed between OCD patients and controls (χ2=8.66, p=0.013). Genotypes were associated with symptom severity (p=0.011), in that patients with the CC genotype had lower Y-BOCS scores than the CT/TT genotypes. While OCD patients diagnosed with MDD had more severe Y-BOCS scores (p=0.023), neither genetic nor epigenetic variation of rs7208505 was associated with comorbid MDD status. Methylation corrected for genotype was not significant in the regression model (p>0.05). Discussion These results provide some evidence that SKA2 genetic variation may be associated with OCD risk and symptom severity. Methylation corrected for genotype was not associated with OCD symptom severity; however, patients with TT and CT genotypes had more severe Y-BOCS scores than the CC genotype carriers. Further work will include replication in larger samples and interrogating additional genetic and epigenetic variations in SKA2.

The Functional Effect of Rare Variants in Complement Genes on C3b Degradation in Patients With Age-Related Macular Degeneration

In age-related macular degeneration (AMD), rare variants in the complement system have been described, but their functional consequences remain largely unexplored. To identify new rare variants in complement genes and determine the functional effect of identified variants on complement levels and complement regulation in serum samples from carriers and noncarriers. This study evaluated affected (n = 114) and unaffected (n = 60) members of 22 families with AMD and a case-control cohort consisting of 1831 unrelated patients with AMD and 1367 control individuals from the European Genetic Database from March 29, 2006, to April 26, 2013, in Nijmegen, the Netherlands, and Cologne, Germany. Exome sequencing data of families were filtered for rare variants in the complement factor H (CFH), complement factor I (CFI), complement C9 (C9), and complement C3 (C3) genes. The case-control cohort was genotyped with allele-specific assays. Serum samples were obtained from carriers of identified variants (n = 177) and age-matched noncarriers (n = 157). Serum concentrations of factor H (FH), factor I (FI), C9, and C3 were measured, and C3b degradation ability was determined. Association of rare variants in the CFH, CFI, C9, and C3 genes with AMD, serum levels of corresponding proteins, and C3b degradation ability of CFH and CFI variant carriers. The 1831 unrelated patients with AMD had a mean (SD) age of 75.0 (9.4) years, and 60.5% were female. The 1367 unrelated control participants had a mean (SD) age of 70.4 (7.0), and 58.7% were female. All individuals were of European descent. Rare variants in CFH, CFI, C9, and C3 contributed to an increased risk of developing AMD (odds ratio, 2.04; 95% CI, 1.47-2.82; P < .001). CFI carriers had decreased median FI serum levels (18.2 μg/mL in Gly119Arg carriers and 16.2 μg/mL in Leu131Arg carriers vs 27.2 and 30.4 μg/mL in noncarrier cases and controls, respectively; both P < .001). Elevated C9 levels were observed in Pro167Ser carriers (10.7 µg/mL vs 6.6 and 6.1 µg/mL in noncarrier cases and controls, respectively; P < .001). The median FH serum levels were 299.4 µg/mL for CFH Arg175Gln and 266.3 µg/mL for CFH Ser193Leu carriers vs 302.4 and 283.0 µg/mL for noncarrier cases and controls, respectively. The median C3 serum levels were 943.2 µg/mL for C3 Arg161Trp and 946.7 µg/mL for C3 Lys155Gln carriers vs 874.0 and 946.7 µg/mL for noncarrier cases and controls, respectively. The FH and FI levels correlated with C3b degradation in noncarriers (R2 = 0.35 and R2 = 0.31, respectively; both P < .001). Reduced serum levels were associated with C3b degradation in carriers of CFI but not CFH variants, suggesting that CFH variants affect functional activity of FH rather than serum levels. Carriers of CFH (Arg175Gln and Ser193Leu) and CFI (Gly119Arg and Leu131Arg) variants have an impaired ability to regulate complement activation and may benefit more from complement-inhibiting therapy than patients with AMD in general.

Transcriptome Sequencing of Diverse Peanut (Arachis) Wild Species and the Cultivated Species Reveals a Wealth of Untapped Genetic Variability.

To test the hypothesis that the cultivated peanut species possesses almost no molecular variability, we sequenced a diverse panel of 22 Arachis accessions representing Arachis hypogaea botanical classes, A-, B-, and K- genome diploids, a synthetic amphidiploid, and a tetraploid wild species. RNASeq was performed on pools of three tissues, and de novo assembly was performed. Realignment of individual accession reads to transcripts of the cultivar OLin identified 306,820 biallelic SNPs. Among 10 naturally occurring tetraploid accessions, 40,382 unique homozygous SNPs were identified in 14,719 contigs. In eight diploid accessions, 291,115 unique SNPs were identified in 26,320 contigs. The average SNP rate among the 10 cultivated tetraploids was 0.5, and among eight diploids was 9.2 per 1000 bp. Diversity analysis indicated grouping of diploids according to genome classification, and cultivated tetraploids by subspecies. Cluster analysis of variants indicated that sequences of B genome species were the most similar to the tetraploids, and the next closest diploid accession belonged to the A genome species. A subset of 66 SNPs selected from the dataset was validated; of 782 SNP calls, 636 (81.32%) were confirmed using an allele-specific discrimination assay. We conclude that substantial genetic variability exists among wild species. Additionally, significant but lesser variability at the molecular level occurs among accessions of the cultivated species. This survey is the first to report significant SNP level diversity among transcripts, and may explain some of the phenotypic differences observed in germplasm surveys. Understanding SNP variants in the Arachis accessions will benefit in developing markers for selection.

High-Throughput Genotyping with TaqMan Allelic Discrimination and Allele-Specific Genotyping Assays.

Real-time PCR-based genotyping methods, such as TaqMan allelic discrimination assays and allele-specific genotyping, are particularly useful when screening a handful of single nucleotide polymorphisms in hundreds of samples; either derived from different individuals, tissues, or pre-amplified DNA. Although real-time PCR-based methods such as TaqMan are well-established, alternative methods, like allele-specific genotyping, are powerful alternatives, especially for genotyping short tandem repeat (STR) length polymorphisms. Here, we describe all relevant aspects when developing an assay for a new SNP or STR using either TaqMan or allele-specific genotyping, respectively, such as primer and probe design, optimization of reaction conditions, the experimental procedure for typing hundreds of samples, and finally the data evaluation. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls involving minimal optimization.