Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio

Air–liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2

The intestinal porcine epithelial cell line IPEC-J2, cultured under the air–liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium–cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response

Prevalence of deleterious germline variants in risk genes including BRCA1/2 in consecutive ovarian cancer patients (AGO-TR-1)

Background: Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated. Methods: Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history. Results: In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16–93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients <60 years was 30.2% (33.2%) versus 10.6% (18.9%) in patients ≥60 years. Family history was positive in 43% of all patients. Patients with a positive family history had a prevalence of deleterious variants of 31.6% (36.0%) versus 11.4% (17.6%) and histologic subtype of high grade serous ovarian cancer versus other showed a prevalence of deleterious variants of 23.2% (29.1%) and 10.2% (14.8%), respectively. Testing only for BRCA1/2 would miss in our series more than 5% of the patients with a deleterious variant in established risk genes. Conclusions: 26.4% of all patients harbor at least one deleterious variant in established risk genes. The threshold of 10% mutation rate which is accepted for reimbursement by health care providers in Germany was observed in all subgroups analyzed and neither age at primary diagnosis nor histo-type or family history sufficiently enough could identify a subgroup not eligible for genetic counselling and testing. Genetic testing should therefore be offered to every patient with invasive epithelial ovarian cancer and limiting testing to BRCA1/2 seems to be not sufficient

Patient characteristics.

<p>Patient characteristics.</p

Histologic subtypes and prevalence of deleterious variants in patients with serous, endometrioid, mucinous or clear cell ovarian cancer.

<p>Histologic subtypes and prevalence of deleterious variants in patients with serous, endometrioid, mucinous or clear cell ovarian cancer.</p

Prevalence of deleterious variants within subgroups.

<p>Prevalence of deleterious variants within subgroups.</p

DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia

Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program

Quantitative Signal Intensity in Fluid-Attenuated Inversion Recovery and Treatment Effect in the WAKE-UP Trial

International audienceBackground and Purpose— Relative signal intensity of acute ischemic stroke lesions in fluid-attenuated inversion recovery (fluid-attenuated inversion recovery relative signal intensity [FLAIR-rSI]) magnetic resonance imaging is associated with time elapsed since stroke onset with higher intensities signifying longer time intervals. In the randomized controlled WAKE-UP trial (Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke Trial), intravenous alteplase was effective in patients with unknown onset stroke selected by visual assessment of diffusion weighted imaging fluid-attenuated inversion recovery mismatch, that is, in those with no marked fluid-attenuated inversion recovery hyperintensity in the region of the acute diffusion weighted imaging lesion. In this post hoc analysis, we investigated whether quantitatively measured FLAIR-rSI modifies treatment effect of intravenous alteplase. Methods— FLAIR-rSI of stroke lesions was measured relative to signal intensity in a mirrored region in the contralesional hemisphere. The relationship between FLAIR-rSI and treatment effect on functional outcome assessed by the modified Rankin Scale (mRS) after 90 days was analyzed by binary logistic regression using different end points, that is, favorable outcome defined as mRS score of 0 to 1, independent outcome defined as mRS score of 0 to 2, ordinal analysis of mRS scores (shift analysis). All models were adjusted for National Institutes of Health Stroke Scale at symptom onset and stroke lesion volume. Results— FLAIR-rSI was successfully quantified in stroke lesions in 433 patients (86% of 503 patients included in WAKE-UP). Mean FLAIR-rSI was 1.06 (SD, 0.09). Interaction of FLAIR-rSI and treatment effect was not significant for mRS score of 0 to 1 ( P =0.169) and shift analysis ( P =0.086) but reached significance for mRS score of 0 to 2 ( P =0.004). We observed a smooth continuing trend of decreasing treatment effects in relation to clinical end points with increasing FLAIR-rSI. Conclusions— In patients in whom no marked parenchymal fluid-attenuated inversion recovery hyperintensity was detected by visual judgement in the WAKE-UP trial, higher FLAIR-rSI of diffusion weighted imaging lesions was associated with decreased treatment effects of intravenous thrombolysis. This parallels the known association of treatment effect and elapsing time of stroke onset

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I 2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None