Performance evaluation of the high-throughput quantitative Alinity m BK virus assay.

BK virus (BKV) infection or reactivation in immunocompromised individuals can lead to adverse health consequences including BKV-associated nephropathy (BKVAN) in kidney transplant patients and BKV-associated hemorrhagic cystitis (BKV-HC) in allogeneic hematopoietic stem cell transplant recipients. Monitoring BKV viral load plays an important role in post-transplant patient care. This study evaluates the performance of the Alinity m BKV Investigational Use Only (IUO) assay. The linearity of the Alinity m BKV IUO assay had a correlation coefficient of 1.000 and precision of SD ≤ 0.25 Log IU/mL for all panel members tested (2.0-7.3 Log IU/mL). Detection rate at 50 IU/mL was 100%. Clinical plasma specimens tested comparing Alinity m BKV IUO to ELITech MGB Alert BKV lab-developed test (LDT) on the Abbott m2000 platform using specimen extraction protocols for DNA or total nucleic acid (TNA) resulted in coefficient of correlation of 0.900 and 0.963, respectively, and mean bias of 0.03 and -0.54 Log IU/mL, respectively. Alinity m BKV IUO compared with Altona RealStar BKV and Roche cobas BKV assays demonstrated coefficient of correlation of 0.941 and 0.980, respectively, and mean bias of -0.47 and -0.31 Log IU/mL, respectively. Urine specimens tested on Alintiy m BKV IUO and ELITech BKV LDT using TNA specimen extraction had a coefficient of correlation of 0.917 and mean bias of 0.29 Log IU/mL. The Alinity m BKV IUO assay was performed with high precision across the dynamic range and correlated well with other available BKV assays. IMPORTANCE: BK virus (BKV) in transplant patients can lead to adverse health consequences. Viral load monitoring is important in post-transplant patient care. This study evaluates the Alinity m BKV assay with currently available assays.

Multicenter performance evaluation of the Alinity m CMV assay for quantifying cytomegalovirus DNA in plasma samples.

Monitoring of cytomegalovirus (CMV) viral load is critical for informing treatment decisions in order to prevent the severe health consequences of CMV infection or reactivation of latent CMV in immunocompromised individuals. This first field evaluation examined the analytical and clinical performance of the Alinity m CMV assay. Analytical performance was assessed with a commercially available six-member panel, while the clinical performance evaluation compared the Alinity m CMV assay to the RealTime CMV assay and a laboratory-developed test (LDT) as the test of record at three large hospital-based clinical laboratories. Precision of the Alinity m CMV assay was demonstrated with total standard deviation (SD) between 0.08 and 0.28 Log IU/mL. A total of 457 plasma specimens were tested on the Alinity m CMV assay and compared to the test of record at each site (n = 304 with RealTime CMV and n = 153 with LDT CMV). The Alinity m CMV assay had excellent correlation (correlation coefficient r ≥0.942) in comparison to the RealTime CMV or LDT CMV assays. The mean observed bias ranged from -0.03 to 0.34 Log IU/mL. Median onboard turnaround time of Alinity m CMV was less than 3 h. When the CMV assay is run on the Alinity m system, it has the capacity to shorten time to result and, therefore, to therapy.

Multi-site performance evaluation of the Alinity m Molecular assay for quantifying Epstein-Barr virus DNA in plasma samples.

Detection and monitoring of acute infection or reactivation of Epstein-Barr virus (EBV) are critical for treatment decision-making and to reduce the risk of EBV-related malignancies and other associated diseases in immunocompromised individuals. The analytical and clinical performance of the Alinity m EBV assay was evaluated at two independent study sites; analytical performance was assessed by evaluating precision with a commercially available 5-member EBV verification panel, while the clinical performance of the Alinity m EBV assay was compared to the RealTime EBV assay and a laboratory-developed test (LDT) as the routine test of record (TOR). Analytical analysis demonstrated standard deviation (SD) between 0.08 and 0.13 Log IU/mL. A total of 300 remnant plasma specimens were retested with the Alinity m EBV assay, and results were compared to those of the TOR at the respective study sites (n = 148 with the RealTime EBV assay and n = 152 with the LDT EBV assay). Agreement between Alinity m EBV and RealTime EBV or LDT EBV assays had kappa values of 0.88 and 0.84, respectively, with correlation coefficients r of 0.956 and 0.912, while the corresponding observed mean bias was -0.02 and -0.19 Log IU/mL. The Alinity m EBV assay had a short median onboard turnaround time of 2:40 h. Thus, the Alinity m system can shorten the time to results and, therefore, to therapy.

The genetics of breast cancer risk in the post-genome era: thoughts on study design to move past BRCA and towards clinical relevance.

More than 12 % of women will be diagnosed with breast cancer in their lifetime. Although there have been tremendous advances in elucidating genetic risk factors underlying both familial and sporadic breast cancer, much of the genetic contribution to breast cancer etiology remains unknown. The discovery of BRCA1 and BRCA2 over 20 years ago remains the seminal event in the field and has paved the way for the discovery of other high-penetrance susceptibility genes by linkage analysis. The advent of genome-wide association studies made possible the next wave of discoveries, in which over 80 low-penetrance and moderate-penetrance variants were identified. Although these studies were highly successful at discovering variants associated with both familial and sporadic breast cancer, the variants identified to date explain only 50 % of the heritability of breast cancer. In this review, we look back at the investigative strategies that have led to our current understanding of breast cancer genetics, consider the challenges of performing association studies in heterogeneous complex diseases such as breast cancer, and look ahead toward the types of study designs that may lead to the identification of the genetic variation accounting for the remaining missing heritability.

Whole-exome Sequence Analysis Implicates Rare Il17REL Variants in Familial and Sporadic Inflammatory Bowel Disease.

BACKGROUND: Rare variants (<1%) likely contribute significantly to risk for common diseases such as inflammatory bowel disease (IBD) in specific patient subsets, such as those with high familiality. They are, however, extraordinarily challenging to identify. METHODS: To discover candidate rare variants associated with IBD, we performed whole-exome sequencing on 6 members of a pediatric-onset IBD family with multiple affected individuals. To determine whether the variants discovered in this family are also associated with nonfamilial IBD, we investigated their influence on disease in 2 large case-control (CC) series. RESULTS: We identified 2 rare variants, rs142430606 and rs200958270, both in the established IBD-susceptibility gene IL17REL, carried by all 4 affected family members and their obligate carrier parents. We then demonstrated that both variants are associated with sporadic ulcerative colitis (UC) in 2 independent data sets. For UC in CC 1: rs142430606 (odds ratio [OR] = 2.99, Padj = 0.028; minor allele frequency [MAF]cases = 0.0063, MAFcontrols = 0.0021); rs200958270 (OR = 2.61, Padj = 0.082; MAFcases = 0.0045, MAFcontrols = 0.0017). For UC in CC 2: rs142430606 (OR = 1.94, P = 0.0056; MAFcases = 0.0071, MAFcontrols = 0.0045); rs200958270 (OR = 2.08, P = 0.0028; MAFcases = 0.0071, MAFcontrols = 0.0042). CONCLUSIONS: We discover in a family and replicate in 2 CC data sets 2 rare susceptibility variants for IBD, both in IL17REL. Our results illustrate that whole-exome sequencing performed on disease-enriched families to guide association testing can be an efficient strategy for the discovery of rare disease-associated variants. We speculate that rare variants identified in families and confirmed in the general population may be important modifiers of disease risk for patients with a family history, and that genetic testing of these variants may be warranted in this patient subset.

Integrated genomic analysis suggests MLL3 is a novel candidate susceptibility gene for familial nasopharyngeal carcinoma.

BACKGROUND: Little is known about genetic factors associated with nasopharyngeal carcinoma (NPC). To gain insight into NPC etiology, we performed whole exome sequencing on germline and tumor DNA from three closely related family members with NPC. METHODS: The family was ascertained through the Pediatric Familial Cancer Clinic at The University of Chicago (Chicago, IL). The diagnosis of NPC was confirmed pathologically for each individual. For each sample sequenced, 97.3% of the exome was covered at 5×, with an average depth of 44×. Candidate germline and somatic variants associated with NPC were identified and prioritized using a custom pipeline. RESULTS: We discovered 72 rare deleterious germline variants in 56 genes shared by all three individuals. Of these, only three are in previously identified NPC-associated genes, all of which are located within MLL3, a gene known to be somatically altered in NPC. One variant introduces an early stop codon in MLL3, which predicts complete loss-of-function. Tumor DNA analysis revealed somatic mutations and Epstein-Barr virus (EBV) integration events; none, however, were shared among all three individuals. CONCLUSIONS: These data suggest that inherited mutations in MLL3 may have predisposed these three individuals from a single family to develop NPC, and may cooperate with individually acquired somatic mutations or EBV integration events in NPC etiology. IMPACT: Our finding is the first instance of a plausible candidate high penetrance inherited mutation predisposing to NPC.

edn1 and hand2 Interact in early regulation of pharyngeal arch outgrowth during zebrafish development.

Endothelin-1 (Edn1) signaling provides a critical input to development of the embryonic pharygneal arches and their skeletal derivatives, particularly the articulating joints and the ventral skeleton including the lower jaw. Previous work in zebrafish has mostly focused on the role of Edn1 in dorsal-ventral (DV) patterning, but Edn1 signaling must also regulate tissue size, for with severe loss of the pathway the ventral skeleton is not only mispatterned, but is also prominently hypoplastic--reduced in size. Here we use mutational analyses to show that in the early pharyngeal arches, ventral-specific edn1-mediated proliferation of neural crest derived cells is required for DV expansion and outgrowth, and that this positive regulation is counterbalanced by a negative one exerted through a pivotal, ventrally expressed Edn1-target gene, hand2. We also describe a new morphogenetic cell movement in the ventral first arch, sweeping cells anterior in the arch to the region where the lower jaw forms. This movement is negatively regulated by hand2 in an apparently edn1-independent fashion. These findings point to complexity of regulation by edn1 and hand2 at the earliest stages of pharyngeal arch development, in which control of growth and morphogenesis can be genetically separated.

FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution.

BACKGROUND: The vertebrate craniofacial skeleton may exhibit anatomical complexity and diversity, but its genesis and evolution can be understood through careful dissection of developmental programs at cellular resolution. Resources are lacking that include introductory overviews of skeletal anatomy coupled with descriptions of craniofacial development at cellular resolution. In addition to providing analytical guidelines for other studies, such an atlas would suggest cellular mechanisms underlying development. DESCRIPTION: We present the Fish Face Atlas, an online, 3D-interactive atlas of craniofacial development in the zebrafish Danio rerio. Alizarin red-stained skulls scanned by fluorescent optical projection tomography and segmented into individual elements provide a resource for understanding the 3D structure of the zebrafish craniofacial skeleton. These data provide the user an anatomical entry point to confocal images of Alizarin red-stained zebrafish with transgenically-labelled pharyngeal arch ectomesenchyme, chondrocytes, and osteoblasts, which illustrate the appearance, morphogenesis, and growth of the mandibular and hyoid cartilages and bones, as viewed in live, anesthetized zebrafish during embryonic and larval development. Confocal image stacks at high magnification during the same stages provide cellular detail and suggest developmental and evolutionary hypotheses. CONCLUSION: The FishFace Atlas is a novel learning tool for understanding craniofacial skeletal development, and can serve as a reference for a variety of studies, including comparative and mutational analyses.

Husbandry of Monodelphis domestica in the study of mammalian embryogenesis.

Monodelphis domestica, commonly called the laboratory opossum, is a useful laboratory animal for studying marsupial embryogenesis and mammalian development. Females breed year-round and the animals can be sustainably bred indoors. The authors draw on their own laboratory's experience to supplement previously published research on laboratory opossums. They describe a breeding protocol that reliably produces timed-pregnant M. domestica. Additionally, the authors discuss general laboratory opossum husbandry techniques and describe how to collect, handle and culture embryos.