Klee, Eric W.

Prior studies in with irritable bowel syndrome with diarrhea (IBS-D) patients showed immune activation, secretion, and barrier dysfunction in jejunal or colorectal mucosa. We measured mRNA expression by RT-PCR of 91 genes reflecting tight junction proteins, chemokines, innate immunity, ion channels, transmitters, housekeeping genes, and controls for DNA contamination and PCR efficiency in small intestinal mucosa from 15 IBS-D and 7 controls (biopsies negative for celiac disease). Fold change was calculated using 2(−ΔΔCT) formula. Nominal P values ( P < 0.05) were interpreted with false detection rate (FDR) correction ( q value). Cluster analysis with Lens for Enrichment and Network Studies (LENS) explored connectivity of mechanisms. Upregulated genes (uncorrected P < 0.05) were related to ion transport (INADL, MAGI1, and SONS1), barrier (TJP1, 2, and 3 and CLDN) or immune functions (TLR3, IL15, and MAPKAPK5), or histamine metabolism (HNMT); downregulated genes were related to immune function (IL-1β, TGF-β1, and CCL20) or antigen detection (TLR1 and 8). The following genes were significantly upregulated ( q < 0.05) in IBS-D: INADL, MAGI1, PPP2R5C, MAPKAPK5, TLR3, and IL-15. Among the 14 nominally upregulated genes, there was clustering of barrier and PDZ domains (TJP1, TJP2, TJP3, CLDN4, INADL, and MAGI1) and clustering of downregulated genes (CCL20, TLR1, IL1B, and TLR8). Protein expression of PPP2R5C in nuclear lysates was greater in patients with IBS-D and controls. There was increase in INADL protein (median 9.4 ng/ml) in patients with IBS-D relative to controls (median 5.8 ng/ml, P > 0.05). In conclusion, altered transcriptome (and to lesser extent protein) expression of ion transport, barrier, immune, and mast cell mechanisms in small bowel may reflect different alterations in function and deserves further study in IBS-D.

Dysferlinopathy is an uncommon, progressive muscular dystrophy that has a wide phenotypic variability and primarily supportive management (Nguyen et al., 2007; Narayanaswami et al., 2014). Amyloid myopathy is a distinct, rare disorder that can present similarly to inflammatory myopathies and requires a high clinical suspicion for early intervention to prolong survival. Amyloid myopathy is typically associated with other systemic manifestations of amyloidosis, but rare cases of isolated amyloid myopathy have been described (Mandl et al., 2000; Hull et al., 2001). Positive Congo red stains on tissue biopsy remain the gold standard for diagnosis (Spuler et al., 1998; Karacostas et al., 2005). A high clinical suspicion and meticulous diagnostic workup that includes novel techniques are necessary for identifying these rare disorders. We report a middle-aged man with progressive leg muscle weakness who was initially treated as having amyloid myopathy but was later diagnosed as having dysferlinopathy by Whole Exome Sequencing (WES) analysis. We also report a novel missense mutation (c.959G>C) to help correlate in any patient with presumed dysferlinopathy and to add to the already known genotype of this disorder.

Abstract BACKGROUND Next generation sequencing (NGS)-based assays continue to redefine the field of genetic testing. Owing to the complexity of the data, bioinformatics has become a necessary component in any laboratory implementing a clinical NGS test. CONTENT The computational components of an NGS-based work flow can be conceptualized as primary, secondary, and tertiary analytics. Each of these components addresses a necessary step in the transformation of raw data into clinically actionable knowledge. Understanding the basic concepts of these analysis steps is important in assessing and addressing the informatics needs of a molecular diagnostics laboratory. Equally critical is a familiarity with the regulatory requirements addressing the bioinformatics analyses. These and other topics are covered in this review article. SUMMARY Bioinformatics has become an important component in clinical laboratories generating, analyzing, maintaining, and interpreting data from molecular genetics testing. Given the rapid adoption of NGS-based clinical testing, service providers must develop informatics work flows that adhere to the rigor of clinical laboratory standards, yet are flexible to changes as the chemistry and software for analyzing sequencing data mature.

Context.— Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α 2 -macroglobulin. Prostate-specific antigen bound to α 2 -macroglobulin is not detected by commercial PSA immunoassays. Objective.— To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays. Design.— Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer. Results.— The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis. Conclusions.— This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays.

Background— Massively parallel, high-throughput sequencing technology is helping to generate new insights into the genetic basis of human diseases. We used whole exome sequencing to identify the mutation underlying a syndrome affecting 2 siblings with aortic hypoplasia, calcific atherosclerosis, systolic hypertension, and premature cataract. Methods and Results— Exonic regions were captured and sequenced using a next-generation sequencing platform to generate 100 bases paired-end reads. A computational genomic data analysis pipeline was used to perform quality control, align reads to a reference genome, and identify genetic variants; findings were confirmed using a different exome analyses pipeline. The 2 siblings were homozygous for a rare missense mutation (Ser818Cys) in INO80D , a subunit of the human INO80 chromatin remodeling complex. Homozygosity mapping and Sanger sequencing confirmed that the mutation is located in one of the runs of homozygosity on chromosome 2. INO80D encodes a key subunit of the human IN080 complex, a multiprotein complex involved in DNA binding, chromatin modification, organization of chromosome structure, and ATP-dependent nucleosome sliding. By introducing a new disulphide-bond in the protein product and also disrupting the composition of low-complexity regions, the Ser818Cys mutation may affect INO80D function, protein–protein interactions, and chromatin remodeling. Conclusions— Our findings suggest a link between the Ser818Cys mutation in INO80D , a subunit of the human INO80 chromatin remodeling complex, and accelerated arterial aging.

BACKGROUNDMultigene molecular testing to guide personalized therapy for oncology patients is of increasing clinical relevance. Molecular testing of fine‐needle aspiration samples is underused, but when acquired with minimally invasive techniques could become the standard of care to obtain theranostic specimens. The aims of the current study were to identify key cytology specimen selection criteria suitable for next‐generation sequencing (NGS) and to determine the prevalence and spectrum of pathogenic alterations in a cohort of patients with AJCC stage IV lung cancer.METHODSA total of 70 adrenal gland cytology specimens with direct smears were screened to identify 56 patients with a single slide containing at least 300 total cells and 20% tumor nuclei. After DNA extraction, the NGS protocols were used to simultaneously detect mutations in >2800 exonic regions in 50 key cancer genes.RESULTSA total of 28 specimens produced acceptable NGS results. Specimens with a combined critical cell mass (>5000 viable cells) and a DNA concentration >5 ng/µL resulted in a 95% chance of successful sequencing. A total of 37 pathogenic alterations were identified in 10 genes and in 25 patients (85%). A pathogenic alteration (≥1) linked to available or developing targeted therapies was revealed in 50% of cases.CONCLUSIONSThe data from the current study demonstrate that theranostic NGS can be applied to adrenal gland metastasis using routine cytologic smear specimens. The characteristics of such smears could be evaluated during onsite adequacy assessment by cytopathology professionals. This model greatly augments the opportunity for customized genotype‐directed therapy from minimally invasive techniques. Cancer (Cancer Cytopathol) 2014;122:822–832. © 2014 American Cancer Society.