Sabine Leh

Ciliopathies with Skeletal Anomalies and Renal Insufficiency due to Mutations in the IFT-A Gene WDR19

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.

Quantitative Contrast-Enhanced Ultrasound Comparison Between Inflammatory and Fibrotic Lesions in Patients with Crohn's Disease

The aim of this study was to determine whether there are differences in absolute blood flow between patients with Crohns disease with inflammation or fibrosis using contrast-enhanced ultrasound. Eighteen patients with fibrotic disease and 19 patients with inflammation were examined. Video sequences of contrast data were analyzed using a pharmacokinetic model to extract the arterial input and tissue residue functions with a custom software, enabling calculation of the absolute values for mean transit time, blood volume and flow. Feasibility of the examination was 89%. The fibrosis group had lower blood volume (0.9 vs. 3.4 mL per 100 mL tissue; p = 0.001) and flow (22.6 vs. 45.3 mL/min per 100 mL tissue; p = 0.003) compared with the inflammation group. There was no significant difference in mean transit time (3.9 vs. 5.5 s). In conclusion, absolute perfusion measurement in the gastrointestinal wall using contrast-enhanced ultrasound is feasible. There seems to be reduced blood volume and blood flow in patients with fibrotic disease.

Validation of Uromodulin as a Candidate Gene for Human Essential Hypertension

A recent genome-wide association study identified a locus on chromosome 16 in the promoter region of the uromodulin (UMOD) gene that is associated with hypertension. Here, we examined the hypertension signal with functional studies in Umod knockout (KO) mice. Systolic blood pressure was significantly lower in KO versus wild-type (WT) mice under basal conditions (KO: 116.6±0.3 mm Hg versus WT: 136.2±0.4 mm Hg; P<0.0001). Administration of 2% NaCl did not alter systolic blood pressure in KO mice, whereas it increased in WT mice by ≈33%, P<0.001. The average 24-hour urinary sodium excretion in the KO was greater than that of WT mice (P<0.001). Chronic renal function curves demonstrate a leftward shift in KO mice, suggesting that the relationship between UMOD and blood pressure is affected by sodium. Creatinine clearance was increased during salt loading with 2% NaCl in the KO mice, leading to augmented filtered Na+ excretion and further Na+ loss. The difference in sodium uptake that exists between WT and KO strains was explored at the molecular level. Urinary tumor necrosis factor-&agr; levels were significantly higher in KO mice compared with WT mice (P<0.0001). Stimulation of primary thick ascending limb of the loop of Henle cells with exogenous tumor necrosis factor-&agr; caused a reduction in NKCC2A expression (P<0.001) with a concurrent rise in the levels of UMOD mRNA (P<0.001). Collectively, we demonstrate that UMOD regulates sodium uptake in the thick ascending limb of the loop of Henle by modulating the effect of tumor necrosis factor-&agr; on NKCC2A expression, making UMOD an important determinant of blood pressure control.

Endorectal elastography in the evaluation of rectal tumours

Aim  Real‐time elastography visualizes tissue compliance using an ultrasound platform. Elastography has been used, particularly in the breast, to characterize indeterminate lesions on B‐mode imaging as either benign or malignant. The primary aim of this study was to assess the feasibility of routine endorectal elastography to evaluate rectal neoplasia. The secondary aim was to correlate elastography data with histopathological end‐points.

Thrombotic microangiopathy mimicking membranoproliferative glomerulonephritis

A 4-year-old boy presented with proteinuria and developed progressive renal failure over 6 years. In the patients family, five individuals were affected with atypical haemolytic uraemic syndrome (aHUS) but not the patient. Renal biopsies (n = 3) showed glomerular basement membrane thickening with double contours, endothelial swelling and deposits of C3 and C1q. Electron microscopy revealed mesangial and subendothelial electron-dense deposits. Complement mutations in membrane cofactor protein (Y155D) and C3 (R713W and G1094R) were detected in all affected family members. The patient also had transient autoantibodies to factor H. The findings suggest that aHUS and glomerulopathy resembling membranoproliferative glomerulonephritis may have a common molecular background.

Mortality in Patients With IgA Nephropathy

BACKGROUND Immunoglobulin A nephropathy (IgAN) is the most common glomerulonephritis globally. Few studies have investigated mortality in patients with IgAN compared with the age- and sex-adjusted general population. STUDY DESIGN Cohort study with record linkage between the Norwegian Kidney Biopsy Registry, Norwegian Cause of Death Registry, and Norwegian Renal Registry. SETTING & PARTICIPANTS 633 patients diagnosed with IgAN in 1988-2004. PREDICTOR Estimated glomerular filtration rate (eGFR), age, and sex. OUTCOMES Deaths and causes of death before and after the onset of end-stage renal disease through 2008. RESULTS Mean follow-up was 11.8 (range, 0-20.8) years. During the observation period, the observed number of deaths was 80 and the expected number was 42.1, resulting in a standardized mortality ratio (SMR) of 1.9 (95% CI, 1.5-2.4). Risk stratification based on initial eGFR showed that SMR was 1.0 (95% CI, 0.6-1.6) if eGFR was ≥60 mL/min/1.73 m(2), 1.9 (95% CI, 1.3-2.8) if eGFR was 30-60 mL/min/1.73 m(2), and 3.6 (95% CI, 2.6-5.0) in patients with eGFR <30 mL/min/1.73 m(2). Renal replacement therapy (RRT) was initiated in 146 patients and 35 of the 80 deaths occurred after the start of RRT. The age- and sex-adjusted SMR was not increased significantly in the pre-RRT period (1.3; 95% CI, 1.0-1.7), but was increased after initiation of RRT (4.9; 95% CI, 3.5-7.0). The most common cause of death was cardiovascular disease, accounting for 45% of all deaths. LIMITATIONS Treatment during follow-up is not known. CONCLUSIONS Mortality in patients with IgAN was twice the expected rate, but not significantly increased before RRT. The risk of end-stage renal disease was substantially higher than risk of death.

Crohn's disease: Comparison of in vitro ultrasonographic images and histology

Objective. To examine some typical histological findings in Crohns disease using high-frequency ultrasound and to define the echo properties of these findings. Material and methods. Bowel resection specimens from 14 patients operated on for Crohns disease were examined with a 10 MHz linear array ultrasound transducer in a saline reservoir. Needles were placed in the specimen corresponding to the ultrasound plane. After formalin fixation, histological sections were taken according to these markings. Fifty-eight ultrasonographic images with 123 regions of interest were compared with corresponding histology. Results. A thickened muscularis mucosae (>0.3 mm) was found in 48 of 69 regions of interest on histology. Submucosa with slight to moderate fibrosis was imaged as an echo-rich layer with sporadic, echo-poor elements (36/56), while severe fibrosis was seen as an echo-rich layer with diffuse, echo-poor elements (40/55). Muscularis propria with slight to moderate fibrosis was seen as an echo-poor layer with sporadic, echo-rich elements (49/66) while severe fibrosis was seen as an echo-poor layer with diffuse, echo-rich elements (17/22). Crohns rosary was seen as echo-poor extensions of the 4th echo layer (31/50). Conclusions. Typical histological findings in Crohns disease such as a thickened muscularis mucosae and Crohns rosary can be imaged with high-frequency ultrasound in vitro. Fibrosis in the submucosa and muscularis propria is associated with decreasing and increasing echogenicity, respectively.

Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.

Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer

Background:The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC).Methods:Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses.Results:Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis.Conclusion:Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Reaccumulation of globotriaosylceramide in podocytes after agalsidase dose reduction in young Fabry patients

Background Agalsidase-α 0.2 mg/kg every other week (eow) and agalsidase-β 1.0 mg/kg/eow are licensed in Europe as equipotent treatment of the α-galactosidase deficiency in Fabry disease. This case series describes the effects of agalsidase dose adjustments in serial kidney biopsies in switch patients. Methods All treatment-naïve patients with classical Fabry disease in our centre started on agalsidase-β 1.0 mg/kg/eow and subsequently switched to agalsidase-α 0.2 mg/kg/eow were included ( n = 3). The median age at enzyme replacement therapy start was 11 (range 7-18) years. Kidney biopsies were performed at baseline, after 5 years of agalsidase-β 1.0 mg/kg/eow and after 3 subsequent years of agalsidase-α 0.2 mg/kg/eow. One patient was re-biopsied 2 years after reswitch to agalsidase-β 1.0 mg/kg/eow. The scoring system of the International Scoring Group of Fabry Nephropathy was used. Results The patients completely cleared globotriaosylceramide (GL3) from mesangial and endothelial cells and partly cleared podocytes on agalsidase-β 1.0 mg/kg/eow. Reaccumulation of GL3 in podocytes, but not in the mesangium or endothelium, occurred after 3 years of agalsidase-α 0.2 mg/kg/eow. Subsequent reduction of podocyte GL3 was observed in the single patient rebiopsied 2 years after reswitch to agalsidase-β 1.0 mg/kg/eow. Conclusion Partial clearance, reaccumulation and renewed partial clearance of podocyte GL3 deposits in serial kidney biopsies over 8-10 years were seen in parallel with agalsidase dose adjustments. Repeated kidney biopsies may impact therapeutic choices in Fabry disease.