Cradic, Kendall W

Introduction Genotype-phenotype discordance occurs occasionally in congenital adrenal hyperplasia (CAH). Its causes are largely unknown. We describe a case of monochorionic, diamniotic twins with discordant clinical presentations of CAH, and show evidence for this being due to mosaicism resulting from a postzygotic full gene deletion of CYP21A2 prior to twinning. Case description A 7-day-old 36-week gestation female infant (Twin A) presented to the emergency department with elevated 17-hydroxyprogesterone (17-OHP). Her identical twin (Twin B) had normal 17-OHP on newborn screening. Both twins showed signs of virilization, more pronounced in Twin B. Molecular genetic testing of both twins and their parents showed a WT paternally-inherited CYP21A2 and a maternally-inherited copy containing the c.293-13C>G mutation. Both twins were also found to have a 5′-CYP21A1P/CYP21A2-3′ hybrid (product of the common 30-kb deletion), derived from the deletion of the paternally-inherited CYP21A2. Neither mother nor father carried the deletion. Conclusions The genetic findings are consistent with mosaicism for two CYP21A2 cell lines in both twins. The first cell line is expected, based on parental results, while the second line is due to a postzygotic full gene deletion of the paternally-inherited WT CYP21A2. The resultant genotype, compound heterozygosity for c.293-13C>G and a CYP21A2 full gene deletion, is consistent with a salt-wasting CAH phenotype. Differential distribution of the second cell line between the twins is most likely the cause for their discrepant phenotypes. We believe this is the first report of somatic CYP21A2 mosaicism, and represents a novel cause for discrepant CAH phenotypes in monozygotic twins.

Abstract Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

Abstract Summary: Mate pair library sequencing is an effective and economical method for detecting genomic structural variants and chromosomal abnormalities. Unfortunately, the mapping and alignment of mate-pair read pairs to a reference genome is a challenging and time-consuming process for most next-generation sequencing alignment programs. Large insert sizes, introduction of library preparation protocol artifacts (biotin junction reads, paired-end read contamination, chimeras, etc.) and presence of structural variant breakpoints within reads increase mapping and alignment complexity. We describe an algorithm that is up to 20 times faster and 25% more accurate than popular next-generation sequencing alignment programs when processing mate pair sequencing. Availability: http://bioinformaticstools.mayo.edu/research/bima/ Contact: vasm@mayo.edu or vasmatzis.george@mayo.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Context: The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. Objective: RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. Design: BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2–3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. Patients: BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. Results: RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. Conclusion: BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.

BACKGROUND The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography–tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold. METHODS After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 μg/L, 25(OH)D2], 2.2 nmol/L [0.9 μg/L, 25(OH)D3], and 10 nmol/L [4 μg/L, 25(OH)D2], 5 nmol/L [2 μg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 μg/L). Interassay CVs across the reportable range were 3.7%–15.2%. Spiked recoveries were 94%–119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n = 1733) and 25(OH)D3 (n = 7614) (R2=0.96 and 0.97), respectively. CONCLUSIONS Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.

Abstract Background: Immunoassays specific for 1–84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. A methodology using immunocapture purification with liquid chromatography–tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope–labeled internal standard, may help address these issues. Methods: We isolated 1–84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1–13 PTH (1SVSEIQLMHNLGK13). Results: The linear range of the assay was 39.1–4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1–44 PTH, 7–84 PTH, 43–68 PTH, 52–84 PTH, 64–84 PTH, and PTH-related protein (PTHrP) was ≤1% to ≤0.001%. Method comparison of LC-MS/MS vs the Roche Cobas® immunoassay yielded Deming fit of LC-MS/MS = 1.01x immunoassay – 13.21. The mean bias by Bland–Altman plot was −9.4%. Conclusions: In patients with hyperparathyroidism, the immunocapture in situ digestion LC-MS/MS method can provide accurate and precise PTH results compared with immunoassay.