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Non-invasive prenatal genetic testing (NIPT) is an advance in the detection of fetal chromosomal aneuploidies that analyzes cell-free DNA (cfDNA) in the blood of a pregnant woman. NIPT has quickly spread across the globe after introduction into clinical practice earlier in this decade. Virtually all professional societies currently recommend that NIPT be used as a screening test rather than a diagnostic method. Due to the extraordinarily high specificity and sensitivity, it is clearly an attractive alternative to conventional serum biochemical screening tests-the current standard of care. We performed a clinical validation study of our Laboratory Developed Test (LDT) assessing equivalency to the verifi™ test by Illumina, Inc. The method uses next generation sequencing (NGS), and features interpretation based on the calculation of normalized chromosomal values (NCV). In our assay, samples from 3334 consecutive patients which had been tested with Illumina’s verifi test, were blinded by an independent third party and retested by the method developed for the Progenity LDT to be offered in our commercial laboratory. As an additional level of validation, we sought and achieved follow up contact on 1681 of the original Illumina cases, to further independently confirm our results and theirs. In the analysis of equivalence between the two tests, 111 trisomy 21 samples, 43 trisomy 18 samples and 21 trisomy 13 samples were identified by both tests, indicating an equivalency of >99.9%. Although the results between the verifi “gold standard” test and our LDT proved identical (i.e., 100% concordance), the verifi test is not promoted by Illumina as having a 100% sensitivity and specificity. Therefore, certain discrepancies can only be appreciated by clinical follow up. In the NCV analyses, categories are created for “affected” or “suspected trisomy” based on the magnitude of the NCV value. Our findings suggest that the very few discrepancies were between the “aneuploidy suspected” categories of the verify reference standard and our LDT’s confirmed “not affected” or “affected” categories. Our conclusion is that NIPT LDTs must be validated by a process including clinical follow-up, not just equivalency to a reference testing method.

SummaryThe complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on “self” result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease.

We report similar to 17.6 million genetic variants from whole-genome sequencing of 2,120 Sardinians; 22% are absent from previous sequencing-based compilations and are enriched for predicted functional consequences. Furthermore, similar to 76,000 variants common in our sample (frequency >5%) are rare elsewhere (

Examining Y The evolution of human populations has long been studied with unique sequences from the nonrecombining, male-specific Y chromosome (see the Perspective by Cann ). Poznik et al. (p. 562 ) examined 9.9 Mb of the Y chromosome from 69 men from nine globally divergent populations—identifying population and individual specific sequence variants that elucidate the evolution of the Y chromosome. Sequencing of maternally inherited mitochondrial DNA allowed comparison between the relative rates of evolution, which suggested that the coalescence, or origin, of the human Y chromosome and mitochondria both occurred approximately 120 thousand years ago. Francalacci et al. (p. 565 ) investigated the sequence divergence of 1204 Y chromosomes that were sampled within the isolated and genetically informative Sardinian population. The sequence analyses, along with archaeological records, were used to calibrate and increase the resolution of the human phylogenetic tree.

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.

e23139Background: Copy number variations and genome-wide DNA hypomethylation represent two characteristics of cancer genome for a variety of cancers. Retrotransposons account for more than 45% of h...

Objective To identify disease-causing mutations within coding regions of 11 known NPHP genes ( NPHP1-NPHP11 ) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost. Methods Mutation analysis was carried out using PCR-based 48.48 Access Array microfluidic technology (Fluidigm) with consecutive next-generation sequencing. We applied a 10-fold primer multiplexing approach allowing PCR-based amplification of 475 amplicons (251 exons) for 48 DNA samples simultaneously. After four rounds of amplification followed by indexing all of 192 patient-derived products with different barcodes in a subsequent PCR, 2×100 paired-end sequencing was performed on one lane of a HiSeq2000 instrument (Illumina). Bioinformatics analysis was performed using ‘CLC Genomics Workbench’ software. Potential mutations were confirmed by Sanger sequencing and shown to segregate. Results Bioinformatics analysis revealed sufficient coverage of 30×for 168/192 (87.5%) DNA samples (median 449×) and of 234 out of 251 targeted coding exons (sensitivity: 93.2%). For proof-of-principle, we analysed 20 known mutations and identified 18 of them in the correct zygosity state (90%). Likewise, we identified pathogenic mutations in 34/192 patients (18%) and discovered 23 novel mutations in the genes NPHP3 (7), NPHP4 (3), IQCB1 (4), CEP290 (7), RPGRIP1L (1), and TMEM67 (1). Additionally, we found 40 different single heterozygous missense variants of unknown significance. Conclusions We conclude that the combined approach of array-based multiplexed PCR-amplification on a Fluidigm Access Array platform followed by next-generation sequencing is highly cost-efficient and strongly facilitates diagnostic mutation analysis in broadly heterogeneous Mendelian disorders.