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The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect. Recently, several publications have shown results on noninvasive detection of MDs by deep sequencing. These approaches demonstrated the proof of concept but are not economically feasible for large-scale clinical applications. We present a novel approach that uses low-coverage whole genome sequencing (approximately 0.2×) to detect MDs genome wide without requiring prior knowledge of the event's location. We developed a normalization method to reduce sequencing noise. We then applied a statistical method to search for consistently increased or decreased regions. A decision tree was used to differentiate whole-chromosome events from MDs. We demonstrated via a simulation study that the sensitivity difference between our method and the theoretical limit was <5% for MDs ≥9 Mb. We tested the performance in a blinded study in which the MDs ranged from 3 to 40 Mb. In this study, our algorithm correctly identified 17 of 18 cases with MDs and 156 of 157 unaffected cases. The limit of detection for any given MD syndrome is constrained by 4 factors: fetal fraction, MD size, coverage, and biological and technical variability of the event region. Our algorithm takes these factors into account and achieved 94.4% sensitivity and 99.4% specificity.

As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA-licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥ 35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result. NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.

Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance. Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13. Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively. These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has developed during the past decade into a versatile tool for biopolymer analysis. The aim of this review is to summarize this development and outline the applications, which have been enabled for routine use in the field of nucleic acid analysis. These include the analysis of mutations, the resequencing of amplicons with a known reference sequence, and the quantitative analysis of gene expression and allelic frequencies in complex DNA mixtures.

Predictive tests for estimating the risk of developing late-stage neovascular age-related macular degeneration (AMD) are subject to unique challenges. AMD prevalence increases with age, clinical phenotypes are heterogeneous and control collections are prone to high false-negative rates, as many control subjects are likely to develop disease with advancing age. Risk prediction tests have been presented previously, using up to ten genetic markers and a range of self-reported non-genetic variables such as body mass index (BMI) and smoking history. In order to maximise the accuracy of prediction for mainstream genetic testing, we sought to derive a test comparable in performance to earlier testing models but based purely on genetic markers, which are static through life and not subject to misreporting. We report a multicentre assessment of a larger panel of single nucleotide polymorphisms (SNPs) than previously analysed, to improve further the classification performance of a predictive test to estimate the risk of developing choroidal neovascular (CNV) disease. We developed a predictive model based solely on genetic markers and avoided inclusion of self-reported variables (eg smoking history) or non-static factors (BMI, education status) that might otherwise introduce inaccuracies in calculating individual risk estimates. We describe the performance of a test panel comprising 13 SNPs genotyped across a consolidated collection of four patient cohorts obtained from academic centres deemed appropriate for pooling. We report on predictive effect sizes and their classification performance. By incorporating multiple cohorts of homogeneous ethnic origin, we obtained >80 per cent power to detect differences in genetic variants observed between cases and controls. We focused our study on CNV, a subtype of advanced AMD associated with a severe and potentially treatable form of the disease. Lastly, we followed a two-stage strategy involving both test model development and test model validation to present estimates of classification performance anticipated in the larger clinical setting. The model contained nine SNPs tagging variants in the regulators of complement activation (RCA) locus spanning the complement factor H ( CFH ), complement factor H-related 4 ( CFHR4 ), complement factor H-related 5 ( CFHR5 ) and coagulation factor XIII B subunit ( F13B ) genes; the four remaining SNPs targeted polymorphisms in the complement component 2 ( C2 ), complement factor B ( CFB ), complement component 3 ( C3 ) and age-related maculopathy susceptibility protein 2 ( ARMS2 ) genes. The pooled sample size (1,132 CNV cases, 822 controls) allowed for both model development and model validation to confirm the accuracy of risk prediction. At the validation stage, our test model yielded 82 per cent sensitivity and 63 per cent specificity, comparable with metrics reported with earlier testing models that included environmental risk factors. Our test had an area under the curve of 0.80, reflecting a modest improvement compared with tests reported with fewer SNPs.

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest.

Abstract Background: Boar taint is an unpleasant odor and flavor of the meat and occurs in a high proportion of uncastrated male pigs. Androstenone, a steroid produced in testis and acting as a sex pheromone regulating reproductive function in female pigs, is one of the main compounds responsible for boar taint. The primary goal of the present investigation was to determine the differential gene expression of selected candidate genes related to levels of androstenone in pigs. Results: Altogether 2560 boars from the Norwegian Landrace and Duroc populations were included in this study. Testicle samples from the 192 boars with most extreme high or low levels of androstenone in fat were used for RNA extraction, and 15 candidate genes were selected and analyzed by real-competitive PCR analysis. The genes Cytochrome P450 c17 (CYP17A1 ), Steroidogenic acute regulatory protein (STAR ), Aldo-keto reductase family 1 member C4 (AKR1C4 ), Short-chain dehydrogenase/reductase family member 4 (DHRS4 ), Ferritin light polypeptide (FTL ), Sulfotransferase family 2A, dehydroepiandrosterone-preferring member 1 (SULT2A1 ), Cytochrome P450 subfamily XIA polypeptide 1 (CYP11A1 ), Cytochrome b5 (CYB5A ), and 17-beta-Hydroxysteroid dehydrogenase IV (HSD17B4 ) were all found to be significantly (P < 0.05) up-regulated in high androstenone boars in both Duroc and Landrace. Furthermore, Cytochrome P450 c19A2 (CYP19A2 ) was down-regulated and progesterone receptor membrane component 1 (PGRMC1 ) was up-regulated in high-androstenone Duroc boars only, while CYP21 was significantly down-regulated (2.5) in high-androstenone Landrace only. The genes Nuclear Receptor co-activator 4 (NCOA4 ), Sphingomyrlin phosphodiesterase 1 (SMPD1 ) and 3β-hydroxysteroid dehydrogenase (HSD3B ) were not significantly differentially expressed in any breeds. Additionally, association studies were performed for the genes with one or more detected SNPs. Association between SNP and androstenone level was observed in CYB5A only, suggesting cis-regulation of the differential transcription in this gene. Conclusion: A large pig material of highly extreme androstenone levels is investigated. The current study contributes to the knowledge about which genes that is differentially expressed regard to the levels of androstenone in pigs. Results in this paper suggest that several genes are important in the regulation of androstenone level in boars and warrant further evaluation of the above mentioned candidate genes, including analyses in different breeds, identification of causal mutations and possible gene interactions.

Biobanks are frequently required to verify specimen relationships. We present two algorithms to compare SNP genotype patterns that provide an objective, high-throughput tool for verification. The first algorithm allows for comparison of all holdings within a biobank, and is well suited to construct sample relationships for comparison with assumed relationships. The second algorithm is tailored to oncology, and allows one to confirm that paired DNAs from malignant and normal tissues are from the same individual in the presence of copy number variations. To evaluate both algorithms, we used an internal training data set (n = 1504) and an external validation data set (n = 1457). In comparison with the results from manual review and knowledge of patient relationships, we identified no errors in interpreting sample relationships within our validation data set. We provide an efficient and objective method of automated data analysis that is currently lacking for establishing and verifying specimen relationships in biobanks. Original submitted 11 October 2012; Revision submitted 25 January 2013

The Peroxisome Poliferator–Activated Receptor-γ2 Pro12Ala Variant Association With Type 2 Diabetes and Trait Differences Julie A. Douglas 1 , Michael R. Erdos 2 , Richard M. Watanabe 3 , Andi Braun 4 , Cristy L. Johnston 4 , Paul Oeth 4 , Karen L. Mohlke 2 , Timo T. Valle 5 , Christian Ehnholm 5 , Thomas A. Buchanan 6 , Richard N. Bergman 7 , Francis S. Collins 2 , Michael Boehnke 1 and Jaakko Tuomilehto 5 8 1 Department of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, Michigan 2 Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, Maryland 3 Division of Biostatistics, Department of Preventative Medicine, Keck School of Medicine, University of Southern California, Los Angeles 4 Sequenom Inc., San Diego, California 5 Department of Epidemiology and Health Promotion, Diabetes and Genetic Epidemiology Unit, and the Department of Biochemistry, National Public Health Institute, Helsinki, Finland 6 Department of Medicine and the 7 Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 8 Department of Public Health, University of Helsinki, Helsinki, Finland Abstract Recent studies have identified a common proline-to-alanine substitution (Pro12Ala) in the peroxisome proliferator–activated receptor-γ2 (PPAR-γ2), a nuclear receptor that regulates adipocyte differentiation and possibly insulin sensitivity. The Pro12Ala variant has been associated in some studies with diabetes-related traits and/or protection against type 2 diabetes. We examined this variant in 935 Finnish subjects, including 522 subjects with type 2 diabetes, 193 nondiabetic spouses, and 220 elderly nondiabetic control subjects. The frequency of the Pro12Ala variant was significantly lower in diabetic subjects than in nondiabetic subjects (0.15 vs. 0.21; P = 0.001). We also compared diabetes-related traits between subjects with and without the Pro12Ala variant within subgroups. Among diabetic subjects, the variant was associated with greater weight gain after age 20 years ( P = 0.023) and lower triglyceride levels ( P = 0.033). Diastolic blood pressure was higher in grossly obese (BMI >40 kg/m 2 ) diabetic subjects with the variant. In nondiabetic spouses, the variant was associated with higher fasting insulin ( P = 0.033), systolic blood pressure ( P = 0.021), and diastolic blood pressure ( P = 0.045). These findings support a role for the PPAR-γ2 Pro12Ala variant in the etiology of type 2 diabetes and the insulin resistance syndrome. AIRG, acute insulin response to glucose dBP, diastolic blood pressure DI, disposition index FUSION, Finland–United States Investigation of Non–Insulin-Dependent Diabetes Mellitus Genetics MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight OGTT, oral glucose tolerance test PPAR, peroxisome proliferator–activated receptor PROBE, primer oligo base extension reaction sBP, systolic blood pressure SI, insulin sensitivity Footnotes Address correspondence and reprint requests to Michael Boehnke, University of Michigan, Department of Biostatistics, 1420 Washington Heights, Ann Arbor, Michigan 48109-2029. E-mail: boehnke{at}umich.edu . Received for publication 12 June 2000 and accepted in revised form 29 December 2000. J.A.D. and M.R.E. contributed equally to this work. Additional information can be found in an online appendix at www.diabetes.org/diabetes/appendix.asp .

Objective As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. Study Design The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA-licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. Results NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age greater than or equal to 35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result. Conclusion NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.