THRB Gene Mosaicism Confirmed by Next-Generation Sequencing in a Clinically Symptomatic Infant.

A 4-day-old infant was admitted for neonatal jaundice. He had persistent tachycardia and tachypnea. Initial workup showed a serum free T4 of 75.6 pmol/L (5.87 ng/dL) (reference range: 11.5-28.3 pmol/L; 0.89-2.20 ng/dL) and a nonsuppressed TSH 3.76 mIU/L (reference range: 0.72-11.0 mIU/L). A TRH stimulation test showed an exaggerated TSH response with a peak of 92.1 mIU/L at 30 minutes after TRH injection, which suggested the diagnosis of resistance to thyroid hormone ß syndrome. Sanger sequencing showed a questionable pathogenic variant in the THRB gene with low signal amplitude. Restriction fragment length polymorphism was consistent with its presence. The variant was originally reported as heterozygous. Next-generation sequencing was performed on blood and buccal swab samples of the patient and his parents, which confirmed this de novo mosaic variant NM_000461.5:c.1352T > C p.(Phe451Ser) in the patient but not in his asymptomatic parents. As it was in a mosaic state, only the offspring, but not other first-degree relatives, of the patient would have the risk of inheriting that variant.

Dopamine infusion at typical infusion rates does not cause interference on plasma creatinine assays.

a Objectives: Dopamine is known to cause negative interference on enzymatic creatinine measurement. However, its effect on the Jaffe reaction, and its concentration required to interfere with enzymatic reactions, remain uncertain. This study was designed to study the interference of stable dopamine infusion on Jaffe and enzymatic creatinine assays, as well as the effect of dopamine infusion drip arm contamination on both creatinine assays. b Design and Methods: For the first part of the study, dopamine was spiked into pooled plasma samples at different concentrations to mimic the scenario of patients on dopamine infusion at an infusion rate between 2 and 20 µg/kg/min. For the second part, dopamine preparation of 2 g/L (same as the preparation used clinically) was mixed with pooled plasma samples at different proportions to mimic drip arm contamination. Creatinine concentrations were measured using Jaffe and enzymatic reactions. c Results: The first part showed that creatinine measurements were not interfered by dopamine infusion at an infusion rate between 2 and 20 µg/kg/min. The second part showed that dopamine could negatively interfere with enzymatic creatinine assays, even with minute drip arm contamination. The effect on the Jaffe reaction was less significant. d Discussion: Creatinine concentration could be reliably measured by Jaffe or enzymatic reactions if samples are from venous access sites other than the site of dopamine infusion. When dopamine interference on enzymatic creatinine assays is suspected, using the Jaffe reaction to cross-check may provide additional useful information.

Vascular Ehlers-Danlos syndrome: A null COL3A1 variant found in a patient with loin pain without marked cutaneous features (case report).

Patients with null variants may have milder vascular Ehlers-Danlos syndrome, presenting with seemingly non-specific complaints and subtle cutaneous features that may be missed. A high index of suspicion and early genetic testing (aided by next-generation sequencing) were crucial for prevention of life-threatening complications in the patient and family members.

GCK-MODY in pregnancy: A pregnant woman with diabetes and a small-for-gestational-age fetus.

Glucokinase-maturity-onset diabetes of the young (GCK-MODY) is often misdiagnosed as other forms of diabetes. A 42-year-old pregnant lady with pre-existing diabetes was treated with insulin during first trimester. Fetal growth restriction was noted since mid-second trimester. Genetic testing suggested the diagnosis of GCK-MODY.

Continuous Glucose Monitoring Metrics in the Assessment of Glycemia in Moderate-to-Advanced CKD in Diabetes.

Introduction: Glycated hemoglobin A1c (HbA1c) has reduced reliability in advanced chronic kidney disease (CKD) owing to factors influencing red cell turnover. Recent guidelines support the use of continuous glucose monitoring (CGM) in glycemic assessment in these patients. We evaluated relationships between HbA1c and CGM metrics of average glycemia and glucose variability (GV) in moderate-to-advanced CKD. Methods: There were a total of 90 patients with diabetes in CKD stages G3b (n = 33), G4 (n = 43), and G5 (nondialysis) (n = 14) (age [mean ± SD] 65.4 ± 9.0 years, estimated glomerular filtration rate [eGFR] 26.1 ± 9.6 ml/min per 1.73 m2, and HbA1c 7.4 ± 0.8%). CGM metrics were estimated from blinded CGM (Medtronic Ipro2 with Enlite sensor) and compared with HbA1c in the same week. Results: Correlations between glucose management indicator (GMI) and HbA1c attenuated with advancing CKD (G3b [r = 0.68, P < 0.0001], G4 [r = 0.52, P < 0.001], G5 [r = 0.22, P = 0.44], P = 0.01 for CKD stage). In G3b and G4, HbA1c correlated significantly with time-in-range (TIR) (3.9-10.0 mmol/l) (r = -0.55 and r = -0.54, respectively) and % time > 13.9 mmol/l (r = 0.53 and r = 0.44, respectively), but not in G5. HbA1c showed no correlation with % time <3.0 mmol/l (r = -0.045, P = 0.67) or % coefficient of variation (CV) (r = -0.05, P = 0.64) in any CKD stage. Only eGFR was a significant determinant of bias for the difference between GMI and HbA1c (difference -0.28%, 95% CI [-0.52 to -0.03] per 15 ml/min per 1.73 m2 decrement, P = 0.03). Conclusion: CGM-derived indices might serve as an adjunct to HbA1c monitoring to guide glycemic management, especially in those with eGFR <30 ml/min per 1.73 m2. Time in hypoglycemia and glycemic variability are relevant glycemic targets for optimization not reflected by HbA1c.

Comprehensive characterization and resolution of discrepant spectrophotometric bilirubin results in patients on eltrombopag therapy.

Background Eltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at ~450 nm (bilirubin), (ii) absorbance at ~550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels. Methods We collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites. Results Forty-nine of 52 samples from patients on ≥50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 µmol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 µmol/L (0.4-3.8 mg/dL) by different routine analyzers, with direct bilirubin ≤4 µmol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r = 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites. Conclusions Eltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

A Hong Kong Chinese kindred with familial hypocalciuric hypercalcaemia caused by AP2S1 mutation.

Familial hypocalciuric hypercalcaemia (FHH) is a genetic disorder of altered calcium homeostasis. Mutations in the CASR, GNA11 and AP2S1 genes have been reported to cause FHH. We report a Hong Kong Chinese kindred with FHH type 3 (FHH3) caused by mutations in AP2S1. The proband, a 51-year-old woman with hypercalcaemia, was initially diagnosed to have primary hyperparathyroidism but repeated parathyroidectomy failed to normalize her plasma calcium concentrations. Later, FHH was suspected and yet no mutations were identified in the CASR gene which causes FHH type 1 (FHH1), the most common form of FHH. Genetic testing of AP2S1 revealed a heterozygous c.43C>T (p.Arg15Cys) mutation, confirming the diagnosis of FHH3. The elder brother and niece of the proband, who both have hypercalcaemia, were found to harbour the same mutation. To our knowledge, this is the first Chinese kindred of FHH3 reported in the English literature.

Hyperandrogenism, Elevated 17-Hydroxyprogesterone and Its Urinary Metabolites in a Young Woman with Ovarian Steroid Cell Tumor, Not Otherwise Specified: Case Report and Review of the Literature.

We describe a case of a 24-year-old overweight woman who presented with hirsutism, secondary amenorrhea, clitoromegaly, and symptoms of diabetes mellitus (DM). While a diagnosis of polycystic ovary syndrome (PCOS) with its associated metabolic disturbances was initially considered, serum total testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) were significantly increased. As 17-OHP did not increase upon ACTH (Synacthen) stimulation and the urinary steroid profile (USP) was compatible with an ovarian source of 17-OHP excess rather than adrenal, non classical congenital adrenal hyperplasia (NCCAH) was unlikely and an androgen-secreting tumor was suspected. Transabdominal ultrasound revealed the presence of an enlarged right ovary with a polycystic ovary morphology and no discrete mass. Transvaginal ultrasound and [18F]- fluorodeoxyglucose positron emission tomography-computed tomography (FDG PET-CT) enabled the localization of a right ovarian tumor. Laparoscopic right salpingo-oophorectomy was performed and a histological diagnosis of steroid cell tumor, not otherwise specified (SCT-NOS) was made. Hyperandrogenism and menstrual disturbances resolved postoperatively. A literature review revealed that 17-OHP-secreting SCT-NOS may uncommonly show positive responses to ACTH stimulation similar to 21-hydroxylase deficiency. Alternatively, USP might be useful in localizing the source of 17-OHP to the ovaries. Its diagnostic performance should be evaluated in further studies.

Epigenomic Promoter Alterations Amplify Gene Isoform and Immunogenic Diversity in Gastric Adenocarcinoma.

Promoter elements play important roles in isoform and cell type-specific expression. We surveyed the epigenomic promoter landscape of gastric adenocarcinoma, analyzing 110 chromatin profiles (H3K4me3, H3K4me1, H3K27ac) of primary gastric cancers, gastric cancer lines, and nonmalignant gastric tissues. We identified nearly 2,000 promoter alterations (somatic promoters), many deregulated in various epithelial malignancies and mapping frequently to alternative promoters within the same gene, generating potential pro-oncogenic isoforms (RASA3). Somatic promoter-associated N-terminal peptides displaying relative depletion in tumors exhibited high-affinity MHC binding predictions and elicited potent T-cell responses in vitro, suggesting a mechanism for reducing tumor antigenicity. In multiple patient cohorts, gastric cancers with high somatic promoter usage also displayed reduced T-cell cytolytic marker expression. Somatic promoters are enriched in PRC2 occupancy, display sensitivity to EZH2 therapeutic inhibition, and are associated with novel cancer-associated transcripts. By generating tumor-specific isoforms and decreasing tumor antigenicity, epigenomic promoter alterations may thus drive intrinsic tumorigenesis and also allow nascent cancers to evade host immunity.Significance: We apply epigenomic profiling to demarcate the promoter landscape of gastric cancer. Many tumor-specific promoters activate different promoters in the same gene, some generating pro-oncogenic isoforms. Tumor-specific promoters also reduce tumor antigenicity by causing relative depletion of immunogenic peptides, contributing to cancer immunoediting and allowing tumors to evade host immune attack. Cancer Discov; 7(6); 630-51. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 539.

Circulating bacterial-derived DNA fragment level is a strong predictor of cardiovascular disease in peritoneal dialysis patients.

BACKGROUND: Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients. METHODS: We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival. RESULTS: The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 - 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005). CONCLUSIONS: Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.

Potential effects of NPC1L1 polymorphisms in protecting against clinical disease in a chinese family with sitosterolaemia.

Sitosterolaemia is caused by mutations in either ABCG5 or ABCG8. Chinese and Japanese individuals usually have mutations in ABCG5. We herein report a known and a novel mutation in ABCG8 and their potential interaction with NPC1L1 polymorphisms in a Chinese family with sitosterolaemia. We sequenced ABCG5 and ABCG8 and measured the levels of plasma plant sterols in a 15-year-old Chinese girl with clinical sitosterolaemia (xanthomas with elevated low-density lipoprotein cholesterol (LDL-C) and plant sterols) and her apparently healthy family members. NPC1L1 was sequenced in the genetically affected sibling and other family members. A known mutation, c.490C＞T (p. Arg164(＊)), in exon 4 and a novel mutation, c.1949T＞G (p.Leu650Arg), in exon 13 of ABCG8 were detected in the proband and her sister, who had elevated sterols but low LDL-C levels and no xanthomas. The genetically affected sister, but not the proband, carried two additional heterozygous changes in NPC1L1 (rs2072183 C＞G, rs2301935 A＞C), which were inherited from the mother, who also had a low LDL-C level. In this study, we detected a known and a novel mutation in ABCG8 in a Chinese patient with sitosterolaemia. The same mutations were found in her clinically normal sister, suggesting that the contrasting features with the proband may be related to different variants in NPC1L1 and/or some other undetermined lipid-related genetic factors.

Early detection of nasopharyngeal carcinoma by plasma Epstein-Barr virus DNA analysis in a surveillance program.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is prevalent in Southeast Asia. Over the last decade, plasma Epstein-Barr virus (EBV) DNA has been developed as a tumor marker for NPC. In this study, the authors investigated whether plasma EBV DNA analysis is useful for NPC surveillance. METHODS: In total, 1318 volunteers ages 40 to 60 years were prospectively recruited. Plasma EBV DNA and serology for viral capsid antigen immunoglobulin A (IgA) were measured. Participants who had detectable plasma EBV DNA or positive IgA serology underwent nasal endoscopic examination and a follow-up plasma EBV DNA analysis in approximately 2 weeks. All participants were followed for 2 years to record the development of NPC. RESULTS: Three individuals with NPC were identified at enrolment. All of them were positive for EBV DNA and remained positive in follow-up analysis. Only 1 of those patients was positive for EBV serology. In 1 patient who had NPC with a small tumor confined to the mucosa, the tumor was not detectable on endoscopic examination. Because of a 2-fold increase in plasma EBV DNA on the follow-up analysis, that patient underwent magnetic resonance imaging, which revealed the tumor. Among the participants who did not have NPC but had initially positive plasma EBV DNA results, approximately 66% had negative EBV DNA results after a median of 2 weeks. CONCLUSIONS: Plasma EBV DNA analysis proved useful for detecting early NPC in individuals without a clinical suspicion of NPC. Repeating the test in those who had initially positive results differentiated those with NPC from those who had false-positive results. Cancer 2013. © 2013 American Cancer Society.

Urinary biomarkers for the prediction of reversibility in acute-on-chronic renal failure.

BACKGROUND: There is no reliable clinical test to predict the reversibility of acute-on-chronic renal failure. We study whether urinary biomarkers could be used as a noninvasive prognostic marker in patients with acute-on-chronic renal failure. METHODS: We studied 39 adult patients with pre-existing chronic renal impairment presenting to us with acute-on-chronic renal failure. Urinary neutrophil gelatinase-associated lipocalin (NGAL) level was measured. The mRNA of kidney injury molecule-1 (KIM-1), interleukin-18 (IL-18), alpha-1-microglobulin (α1M), sodium/hydrogen exchanger-3 (NHE3), beta-2 microglobulin (ß2M), and N-acetyl-ß-D-glucosaminidase (NAG) in urinary sediment were quantified. RESULTS: Urinary NGAL level significantly correlated with the serum creatinine at presentation (r=0.762, p<0.0001) but not baseline serum creatinine. Urinary sediment ß2M expression significantly correlated with baseline glomerular filtration rate (GFR) (r=−0.400, p=0.012). Urinary α1M and NHE3 expressions were significantly higher in ischemic acute tubular necrosis than other causes of acute kidney injury (p<0.0001 and p=0.006, respectively). Urinary α1M expression significantly correlated with the degree of improvement in renal function (r=0.387, p=0.026), as well as the estimated GFR 6 months later (r=0.386, p=0.027). CONCLUSION: In patients with acute-on-chronic renal failure, urinary NGAL level correlates with the severity of renal failure, while urinary α1M expression correlates with the degree of renal function recovery. Quantification of urinary α1M mRNA may be developed as an non-invasive tool for risk stratification of this group of patients.

Bromodomain-containing protein 4 (BRD4) regulates RNA polymerase II serine 2 phosphorylation in human CD4+ T cells.

Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser-2-phosphorylated Pol II (Pol II Ser-2) at both enhancers and promoters of active genes. Disruption of bromodomain-histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser-2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited diacetylated H4 (lysine 5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells.