Jörg Bojunga

Common genetic determinants of vitamin D insufficiency: a genome-wide association study

BACKGROUND Vitamin D is crucial for maintenance of musculoskeletal health, and might also have a role in extraskeletal tissues. Determinants of circulating 25-hydroxyvitamin D concentrations include sun exposure and diet, but high heritability suggests that genetic factors could also play a part. We aimed to identify common genetic variants affecting vitamin D concentrations and risk of insufficiency. METHODS We undertook a genome-wide association study of 25-hydroxyvitamin D concentrations in 33 996 individuals of European descent from 15 cohorts. Five epidemiological cohorts were designated as discovery cohorts (n=16 125), five as in-silico replication cohorts (n=9367), and five as de-novo replication cohorts (n=8504). 25-hydroxyvitamin D concentrations were measured by radioimmunoassay, chemiluminescent assay, ELISA, or mass spectrometry. Vitamin D insufficiency was defined as concentrations lower than 75 nmol/L or 50 nmol/L. We combined results of genome-wide analyses across cohorts using Z-score-weighted meta-analysis. Genotype scores were constructed for confirmed variants. FINDINGS Variants at three loci reached genome-wide significance in discovery cohorts for association with 25-hydroxyvitamin D concentrations, and were confirmed in replication cohorts: 4p12 (overall p=1.9x10(-109) for rs2282679, in GC); 11q12 (p=2.1x10(-27) for rs12785878, near DHCR7); and 11p15 (p=3.3x10(-20) for rs10741657, near CYP2R1). Variants at an additional locus (20q13, CYP24A1) were genome-wide significant in the pooled sample (p=6.0x10(-10) for rs6013897). Participants with a genotype score (combining the three confirmed variants) in the highest quartile were at increased risk of having 25-hydroxyvitamin D concentrations lower than 75 nmol/L (OR 2.47, 95% CI 2.20-2.78, p=2.3x10(-48)) or lower than 50 nmol/L (1.92, 1.70-2.16, p=1.0x10(-26)) compared with those in the lowest quartile. INTERPRETATION Variants near genes involved in cholesterol synthesis, hydroxylation, and vitamin D transport affect vitamin D status. Genetic variation at these loci identifies individuals who have substantially raised risk of vitamin D insufficiency. FUNDING Full funding sources listed at end of paper (see Acknowledgments).

Severe lactic acidosis during treatment of chronic hepatitis B with entecavir in patients with impaired liver function

Entecavir is a potent nucleoside inhibitor of the hepatitis B virus (HBV) polymerase with a high antiviral efficacy and a high genetic barrier to viral resistance. After approval in 2006, knowledge on the side effect profile in patients with advanced liver disease and impaired liver function is still limited. Here, we report on 16 patients with liver cirrhosis and chronic hepatitis B who were treated with entecavir. Five of these patients developed lactic acidosis during entecavir treatment. All patients who developed lactic acidosis had highly impaired liver function (Model for End‐Stage Liver Disease [MELD] score ≥ 20). Lactic acidosis (lactate 26‐200 mg/dL, pH 7.02‐7.40, base excess −5 mmol/L to −18 mmol/L) occurred between 4 and 240 days after treatment initiation with entecavir. Lactic acidosis was lethal in one patient but resolved in the other cases after termination/interruption of entecavir treatment. No increased lactate serum concentrations were observed during treatment with entecavir in the other 11 patients with chronic hepatitis B and liver cirrhosis who all had MELD scores below 18. The MELD score correlated with the development of lactic acidosis (P < 0.005) as well as its single parameters bilirubin, international normalized ratio, and creatinine. In contrast, Child‐Pugh Score did not correlate with the development of lactic acidosis. Our data indicate that entecavir should be applied cautiously in patients with impaired liver function. (HEPATOLOGY 2009;50:2001–2006.)

Vitamin D deficiency and a CYP27B1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy

BACKGROUND & AIMS Vitamin D is an important immune modulator and preliminary data indicated an association between vitamin D deficiency and sustained virologic response (SVR) rates in hepatitis C virus (HCV) genotype 1 patients. We, therefore, performed a comprehensive analysis on the impact of vitamin D serum levels and of genetic polymorphisms with functional relevance within the vitamin D cascade on chronic hepatitis C and its treatment. METHODS Vitamin D serum levels, genetic polymorphisms within the vitamin D receptor and 1α-hydroxylase were determined in a cohort of 468 HCV genotype 1, 2, and 3 infected patients who were treated with interferon-alfa based regimens. RESULTS Chronic hepatitis C was associated with a high incidence of severe vitamin D deficiency compared to controls (25(OH)D(3)<10 ng/ml in 25% versus 12%, p<0.00001). 25(OH)D(3) deficiency correlated with SVR in HCV genotype 2 and 3 patients (50% and 81% SVR for patients with and without severe vitamin D deficiency, respectively, p<0.0001). In addition, the CYP27B1-1260 promoter polymorphism rs10877012 had substantial impact on 1,25-dihydroxyvitamin D serum levels (72, 61, and 60 pmol/ml for rs10877012 AA, AC, and CC, respectively, p=0.04) and on SVR rates in HCV genotype 1, 2, and 3 infected patients (77% and 65% versus 42% for rs10877012 AA, AC, and CC, respectively, p=0.02). CONCLUSIONS Chronic hepatitis C virus infection is associated with vitamin D deficiency. Reduced 25-hydroxyvitamin D levels and CYP27B1-1260 promoter polymorphism leading to reduced 1,25-dihydroxyvitamin D levels are associated with failure to achieve SVR in HCV genotype 1, 2, and 3 infected patients.

Real-time Elastography and Contrast-Enhanced Ultrasound for the Assessment of Thyroid Nodules

OBJECTIVE Work-up of thyroid nodules remains challenging. Recent technologies enable determination of tissue elasticity and perfusion using ultrasound devices. The aim of the present study was to evaluate real-time elastography (RTE) and contrast-enhanced ultrasound with Sonovue (CEUS) for the differentiation of benign and malignant thyroid nodules. MATERIALS AND METHODS Inclusion criteria were: nodules ≥1 cm, non-functioning or hypo-functioning on radionuclide scanning, and cytological/histological assessment. All patients received conventional ultrasound, RTE and CEUS. RTE was classified as: Elasticity-Score (ES)1 = soft, ES2 = predominantly soft, ES3 = predominantly hard, ES4 = hard nodule. CEUS-video clips were digitally recorded and analyzed using time-intensity-curves within selected regions-of-interest. RESULTS Fifty-three nodules in 50 patients were available for analysis. Forty-six nodules were benign on cytology/histology, 6 nodules were papillary carcinoma and one nodule was a follicular carcinoma. Nodule margin irregularity was the ultrasound pattern most predictive of malignancy with sensitivity 57% (95% confidence interval: 18-90%) and specificity 85% (71-94% p<0.05). When using ES3&4 for the diagnosis of malignant nodules sensitivity and specificity were 86% (42-99.7%) and 87% (75-95%), respectively (p = 0.0003). The only malignant nodule missed with RTE was a follicular carcinoma. Sensitivity for the diagnosis of papillary carcinoma therefore was 100%. No specific CEUS pattern could be identified to differentiate between benign and malignant nodules. CONCLUSIONS RTE seems to be a useful tool in the work-up of thyroid nodules to exclude papillary thyroid cancer. However, follicular carcinoma remains a challenging problem. CEUS did not improve the characterization of thyroid nodules in this preliminary study.

Molecular detection of thyroglobulin mRNA transcripts in peripheral blood of patients with thyroid disease by RT-PCR

The sensitive detection of circulating tumour cells in patients with differentiated thyroid cancer may precede the detection of relapse by other diagnostic studies – such as serum thyroglobulin – and thus may have important therapeutic and prognostic implications. We performed reverse transcription-polymerase chain reaction (RT-PCR) on blood samples from patients diagnosed with thyroid disease using two different RT-PCR sensitivities. Additionally, tissue specificity of TG mRNA-expression was determined using RNA extracts from 27 different human tissues. The lower limit of detection was 50–100 TG mRNA producing cells/ml blood using a ‘normal’ RT-PCR sensitivity and 10–20 cells/ml blood using a ‘high’ sensitivity. With the normal sensitivity TG mRNA was detected in 9/13 patients with thyroid cancer and metastasis, 63/137 patients with a history of thyroid cancer and no metastasis, 21/85 with non-malignant thyroid disease and 9/50 controls. With the high sensitivity TG mRNA was detected in 11/13 patients with thyroid cancer and metastasis, 111/137 patients with a history of thyroid cancer and no metastasis, 61/85 with non-malignant thyroid disease and 41/50 controls. Interestingly, using the normal RT-PCR sensitivity TG mRNA transcripts are specific for thyroid tissue and detectable in the peripheral blood of controls and patients with thyroid disease, which correlates with a diagnosis of metastasized thyroid cancer. However, with a high RT-PCR sensitivity, TG mRNA expression was found not to be specific for thyroid tissue and was not correlated with a diagnosis of thyroid cancer in patients. As a consequence, to date TG mRNA detected by RT-PCR in the peripheral blood cannot be recommended as a tumour marker superior to TG serum-level.

Low vitamin D serum concentration is associated with high levels of hepatitis B virus replication in chronically infected patients

Vitamin D is an important immune modulator that plays an emerging role in inflammatory and metabolic liver diseases, including infection with hepatitis C virus (HCV). In contrast, the relationship between vitamin D metabolism and chronic hepatitis B (CHB) is less well characterized. Therefore, we quantified 25(OH)D3 serum levels in a cohort of 203 treatment‐naïve patients with chronic hepatitis B virus (HBV) infection and tested for their association with clinical parameters of CHB. Of 203 patients, 69 (34%), 95 (47%), and 39 (19%) had severe vitamin D deficiency (25(OH)D3 <10 ng/mL), vitamin D insufficiency (25(OH)D3 ≥10 and <20 ng/mL), or adequate vitamin D serum levels (25(OH)D3 ≥20 ng/mL), respectively. In both uni‐ and multivariate analyses, HBV DNA viral load (log10 IU/mL) was a strong predictor of low 25(OH)D3 serum levels (P = 0.0007 and P = 0.000048, respectively) and vice versa. Mean 25(OH)D3 serum concentrations in patients with HBV DNA <2,000 versus ≥2,000 IU/mL were 17 versus 11 ng/mL, respectively (P < 0.00001). In addition, hepatitis B early antigen (HBeAg)‐positive patients had lower 25(OH)D3 serum levels than HBeAg‐negative patients (P = 0.0013). Finally, 25(OH)D3 and HBV DNA serum levels showed inverse seasonal fluctuations. Conclusion: Low 25(OH)D3 serum levels are associated with high levels of HBV replication in patients with CHB. This represents a major difference from chronic hepatitis C, where numerous previous studies have shown a lack of correlation between HCV viral load and vitamin D serum levels. Inverse seasonal fluctuations of 25(OH)D3 and HBV DNA serum levels are suggestive of a functional relationship between both variables. (Hepatology 2013;58:1270–1276)

Real-Time Tissue Elastography Versus FibroScan for Noninvasive Assessment of Liver Fibrosis in Chronic Liver Disease

PURPOSE Transient elastography (FibroScan, [TE]) and serum fibrosis markers such as the FibroTest (FT) are established methods for the noninvasive staging of liver fibrosis. A study using real-time elastography (HI-RTE), which is integrated in a conventional ultrasound system, was recently published with comparable results to transient elastography. The aim of the present study was to validate real-time elastography using the formulas calculated in previous studies and to compare the results to transient elastography and FibroTest for the noninvasive assessment of liver fibrosis. MATERIALS AND METHODS One hundred and thirty-four patients with chronic liver disease and either histological assessment of liver fibrosis (n = 112) or proven liver cirrhosis (n = 22) were included in the study. All patients received TE, HI-RTE, and biochemical evaluation on the same day as presentation. The calculation of the elasticity score of real-time elastography was performed in accordance with the two previously published studies. RESULTS The Spearman correlation coefficient between transient elastography, real-time elastography and FibroTest with the histological Chevallier score was statistically significant with 0.78, 0.34, and 0.67, respectively (p < 0.01). The diagnostic accuracy expressed as areas under ROC curves was 0.84, 0.69 and 0.85 for the diagnosis of significant fibrosis (F > or = 2), and 0.97, 0.65, and 0.83 for the diagnosis of cirrhosis, respectively. CONCLUSION Real-time elastography in its present form cannot replace transient elastography for noninvasive assessment of liver fibrosis.

Comparison of acoustic radiation force impulse imaging with transient elastography for the detection of complications in patients with cirrhosis

Acoustic radiation force impulse (ARFI) imaging is a new non‐invasive, ultrasound‐based method for the evaluation of liver fibrosis and cirrhosis.

A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C

Background To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C. Methodology/Principal Findings Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D3 (25[OH]D3) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D3<20 ng/mL) during all seasons, but 25(OH)D3 serum levels were not associated with treatment outcome. Conclusions/Significance Our study suggests a role of bioactive vitamin D (1,25[OH]2D3, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D3 is not a suitable predictor of treatment outcome.

Soluble ICAM-1 reduces leukocyte adhesion to vascular endothelium in ischemia-reperfusion injury in mice.

Ischemia-reperfusion injury is a pathogenic factor in the course of many clinical disorders, such as myocardial infarction, stroke, organ transplantation, burns, and circulatory shock. The extent of ischemia-reperfusion injury is dependent on the number of infiltrating leukocytes. With in vivo microscopy, we evaluated the effect of the recombinant form of soluble murine intercellular adhesion molecule-1 (ICAM-1) on ischemia-reperfusion injury in an animal model. A mesenteric vein was occluded with a clamp for 45 min. During a reperfusion period of 30 min, the number of leukocytes rolling along the endothelium and the number of adherent leukocytes were measured with and without pretreatment with recombinant ICAM-1. The number of leukocytes rolling along the endothelial surface increased more than twofold during postischemic perfusion ( P < 0.05). Recombinant ICAM-1 had no effect on leukocyte rolling. In the control group, firm adherence of leukocytes was increased 10-fold. Recombinant ICAM-1 dose dependently reduced firm adhesion to the endothelium in response to prior ischemia. After 30 min, reperfusion pretreatment with recombinant ICAM-1 inhibited leukocyte adherence from 512 ± 123 to 166 ± 34 leukocytes/mm2( P < 0.01). We demonstrate here for the first time that soluble recombinant ICAM-1 is able to reduce leukocyte adherence to mesenteric venules in postischemic reperfusion injury dose dependently. Because soluble ICAM-1 is naturally circulating in human serum, the therapeutic use of soluble recombinant forms of ICAM-1 may represent a physiological way to protect against ischemiareperfusion injury.Ischemia-reperfusion injury is a pathogenic factor in the course of many clinical disorders, such as myocardial infarction, stroke, organ transplantation, burns, and circulatory shock. The extent of ischemia-reperfusion injury is dependent on the number of infiltrating leukocytes. With in vivo microscopy, we evaluated the effect of the recombinant form of soluble murine intercellular adhesion molecule-1 (ICAM-1) on ischemia-reperfusion injury in an animal model. A mesenteric vein was occluded with a clamp for 45 min. During a reperfusion period of 30 min, the number of leukocytes rolling along the endothelium and the number of adherent leukocytes were measured with and without pretreatment with recombinant ICAM-1. The number of leukocytes rolling along the endothelial surface increased more than twofold during postischemic perfusion (P < 0.05). Recombinant ICAM-1 had no effect on leukocyte rolling. In the control group, firm adherence of leukocytes was increased 10-fold. Recombinant ICAM-1 dose dependently reduced firm adhesion to the endothelium in response to prior ischemia. After 30 min, reperfusion pretreatment with recombinant ICAM-1 inhibited leukocyte adherence from 512 +/- 123 to 166 +/- 34 leukocytes/mm2 (P < 0.01). We demonstrate here for the first time that soluble recombinant ICAM-1 is able to reduce leukocyte adherence to mesenteric venules in postischemic reperfusion injury dose dependently. Because soluble ICAM-1 is naturally circulating in human serum, the therapeutic use of soluble recombinant forms of ICAM-1 may represent a physiological way to protect against ischemiareperfusion injury.