Prospective evaluation of the RT-PCR based urinary marker Bladder Epicheck® as a diagnostic tool in upper urinary tract tumor.

BACKGROUND: Upper-tract-urothelial-carcinoma (UTUC) represents 5-10% of all urothelial-neoplasms with increasing incidence in the last decades. Current standard tools for diagnosis of UTUC include cytology, computed tomography (CT) urography and ureterorenoscopy (URS). The aim of this study was to evaluate the impact of Bladder Epicheck® Test as diagnostic tool for UTUC diagnosis and recurrence. METHODS: Overall, 136 urine samples, selective collected from upper-urinary-tract before URS for suspicion of UTUC were analyzed with cytology and Bladder Epicheck® Test. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of both markers were calculated and compared to URS and/or histology as reference. RESULTS: UTUC was detected in 40 cases (33.3%), among them 30 were classified as low-grade (LG) and 10 as high-grade (HG). Overall sensitivity of Bladder Epicheck® for UTUC detection was 65% compared to 42.5% for cytology, increasing to 100% for Bladder Epicheck® and 90% for cytology if considering only HG tumors. Overall specificity of Bladder Epicheck® was 81.2% and of cytology 93.7%. PPV and NPV were 63.4% and 82.2% for Bladder Epicheck® and 77.2% and 76.5% for cytology. Considering an EpiScore cut-off >75, instead of 60, specificity of Bladder Epicheck® improves to 89% and PPV to 74.2%. Limitations include the use of a marker validated only for bladder-cancer and the relatively small number of cases. CONCLUSIONS: Due to its high sensitivity for HG tumors, the Bladder Epicheck® Test can be used in diagnosis and treatment decision-making of UTUC. Furthermore, it could be very useful in follow-up of UTUC, after endoscopic treatment to postpone or avoid unnecessary endoscopic exploration. Even if further studies are needed to validate these findings, Bladder Epicheck® could be a promising clinical tool for detection of UTUC.

Evaluation of the M371-Test Under Real-life Conditions for Diagnosis and Follow Up of Testicular Germ Cell Tumors.

BACKGROUND/AIM: The aim of the study was to establish the performance of the M371-Test on the Thermocycler Rotor-GeneQ (Qiagen) platform for diagnosis and follow-up of testicular tumors and to evaluate the test under real-life conditions in comparison to the classical markers alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (ß-HCG) and lactate dehydrogenase (LDH). PATIENTS AND METHODS: Forty-four patients, of median age 29 years (range=24-84) were included in this prospective study at our institution between March 2021 and September 2022. Of the 44 patients, 23 had a suspicion of testicular cancer (TC) and 21 were under follow-up for TC. In total, 96 M371-Tests were performed and compared with AFP, ß-HCG, LDH using histological diagnosis and/or computer tomography (CT) scan as the gold standard. RESULTS: In the patients with suspicion of TC, the M371-Test showed a sensitivity of 73.7%, AFP of 21%, LDH of 31.6% and ß-HCG of 42.1%. In the patients under follow-up for TC, the M371-Test showed a sensitivity of 86.4%, AFP of 50%, LDH of 31.8% and ß-HCG of 63.6%. In germ cell tumours (GCT)/non-seminomas, M371-Test had a sensitivity of 83.3%, AFP of 77.8%, LDH of 38.9% and ß-HCG of 66.7%. In GCT/seminomas, M371-Test had a sensitivity of 85%, AFP of 5%, LDH of 30% and ß-HCG of 50%. CONCLUSION: Under real life conditions performed on the real-time Thermocycler Rotor-GeneQ (Qiagen) platform, the M371-Test shows an outstanding performance and is far beyond the sensitivity of the classical markers for detecting GCTs and in the follow-up of patients after GCT, especially in seminomas.

Diagnostic value of Xpert® BC Detection, Bladder Epicheck®, Urovysion® FISH and cytology in the detection of upper urinary tract urothelial carcinoma.

PURPOSE: Following the current guidelines, diagnosis and staging for upper urinary tract tumours (UTUC) can be performed with Computed Tomography, urography, ureterorenoscopy (URS) and selective cytology. The aim of the study was to evaluate the performance of the Xpert®-BC-Detection and the Bladder-Epicheck®-test in the detection of UTUC and compare it with cytology and the Urovysion®-FISH test using histology and URS as gold standard. METHODS: A total of 97 analyses were collected through selective catheterization of the ureter before URS to test for cytology, Xpert®-BC-Detection, Bladder-Epicheck® and Urovysion®-FISH. Sensitivity, specificity, and predictive values were calculated using histology results/URS as reference. RESULTS: Overall sensitivity was 100% for Xpert®-BC-Detection, 41.9% for cytology, 64.5% for Bladder-Epicheck® and 87.1% for Urovysion®-FISH. The sensitivity of Xpert®-BC-Detection was 100% in both, LG and HG tumours, sensitivity of cytology increased from 30.8% in LG to 100% in HG, for Bladder-Epicheck® from 57.7% in LG to 100% in HG and of Urovysion®-FISH from 84.6% in LG to 100% in HG tumours. Specificity was 4.5% for Xpert®-BC-Detection, 93.9% for cytology, 78.8% for Bladder-Epicheck® and 81.8% for Urovysion®-FISH. PPV was 33% for Xpert®-BC-Detection, 76.5% for cytology, 58.8% for Bladder-Epicheck® and 69.2% for Urovysion®FISH. NPV was 100% for Xpert®-BC-Detection, 77.5% for cytology, 82.5% for Bladder-Epicheck® and 93.1% for Urovysion®FISH. CONCLUSION: Bladder-Epicheck® and Urovysion®FISH along with cytology could be a helpful ancillary method in the diagnosis and follow-up of UTUC while due to its low specificity Xpert®-BC Detection seems to be of limited usefulness.

Induced Pluripotent Stem Cell (iPSC) Lines from a Family with Resistant Epileptic Encephalopathy Caused by Compound Heterozygous Mutations in SZT2 Gene.

Mutations in the SZT2 gene have been associated with developmental and epileptic encephalopathy-18, a rare severe autosomal recessive neurologic disorder, characterized by psychomotor impairment/intellectual disability, dysmorphic facial features and early onset of refractory seizures. Here we report the generation of the first induced pluripotent stem cell (iPSC) lines from a patient with treatment-resistant epilepsy, carrying compound heterozygous mutations in SZT2 (Mut1: c.498G>T and Mut2: c.6553C>T), and his healthy heterozygous parents. Peripheral blood mononuclear cells were reprogrammed by a non-integrating Sendai virus-based reprogramming system. The generated human iPSC lines exhibited expression of the main pluripotency markers, the potential to differentiate into all three germ layers and presented a normal karyotype. These lines represent a valuable resource to study neurodevelopmental alterations, and to obtain mature, pathology-relevant neuronal populations as an in vitro model to perform functional assays and test the patient's responsiveness to novel antiepileptic treatments.

Xpert® bladder cancer detection as a diagnostic tool in upper urinary tract urothelial carcinoma: preliminary results.

Objectives: Upper urinary tract urothelial carcinoma (UTUC) represents about 5-10% of all urothelial malignancies with an increasing incidence. The standard diagnostic tools for the detection of UTUC are cytology, computed tomography (CT) urography, and ureterorenoscopy (URS). No biomarker to be included in the daily clinical practice has yet been identified. The aim of our study was to evaluate the potential role of Xpert® Bladder-Cancer (BC)-Detection in the diagnosis of UTUC. Methods: Eighty-two patients underwent 111 URS with Xpert® BC-Detection, cytology, or Urovysion® analysis of UT for suspicion of UTUC. Twenty-four cases were excluded from the analysis due to a non-diagnostic Xpert® BC-Detection, cytology, or Urovysion®. Samples were analyzed with upper tract (UT) urinary cytology, with Xpert® BC-Detection on UT urines, and with Urovysion® Fluorescence in situ hybridization (FISH) test. After urine collection, the patients underwent retrograde pyelography and/or URS, and if positive a UT biopsy. The Xpert® BC-Detection was reported by the software as negative or positive [cut-off total Linear Discriminant Analysis (LDA) = 0.45]. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of cytology, Xpert® BC-Detection and Urovysion-FISH were calculated using URS and/or histology results as reference. Results: In all, 27 (31%) of 87 URS resulted positive, with 20 low-grade (LG) and 7 high-grade (HG) tumors. Overall sensitivity was 51.9% for cytology, 100% for Xpert® BC-Detection, and 92.6% for Urovysion. The sensitivity of cytology increased from 26% in LG to 100% in HG tumors. For Xpert® BC-Detection, sensitivity was 100% both in LG and in HG, and for Urovysion-FISH, it increased from 90% in LG to 100% in HG tumors. PPV was 82.4% for cytology, 35% for Xpert® BC-Detection, and 73.5% for Urovysion. NPV was 81.4% for cytology, 100% for Xpert® BC-Detection, and 96.2% for Urovysion. Conclusion: The excellent NPV of Xpert® BC-Detection allows to avoid unnecessary endoscopic exploration of the UT, reducing invasiveness and URS complications in the follow-up of UTUC.

Exome-wide association study of levodopa-induced dyskinesia in Parkinson's disease.

Levodopa is the standard long-term dopamine replacement therapy to treat Parkinson's disease (PD) symptoms. With time, levodopa may induce debilitating dyskinesias (LID), the treatment of which represents a large clinically unmet need. However, time-to-LID onset varies between patients, reflecting a possible genetic component. We performed an hypothesis-free whole-exome sequencing (WES)-based screening of time-to-LID onset and attempted replication of previously published candidate gene studies. A WES association analysis was carried out in 134 PD patients in a meta-analytical framework. Replication was attempted in an independent study of 97 PD patients. Variants from previously reported candidate genes (OPRM1, COMT, BDNF) were also specifically examined. We significantly replicated, for the first time, an association of variant rs1799971 in the OPRM1 gene with time-to-LID onset. Furthermore, we identified two novel potentially functional variants, in the MAD2L2 (rs2233019) and MAP7 (rs35350783) genes, which were significantly associated at the discovery stage. In the replication study, the two variants showed direction-consistent effects but did not achieve the replication significance threshold. Our study provides the first WES results for time-to-LID onset, where we replicate association at OPRM1, and suggest new variants in MAD2L2 and MAP7 genes that are significant in discovery, but require larger datasets for replication. The results are being made publicly available to allow for independent external validation.

Diagnostic value of Xpert® Bladder Cancer Monitor in the follow-up of patients affected by non-muscle invasive bladder cancer: an update.

AIMS: Xpert® Bladder Cancer Monitor is a urinary marker based on the evaluation of five target mRNAs overexpressed in patients with bladder cancer (BC). The aim of our study is to update our results regarding the diagnostic accuracy of the Xpert® Bladder Cancer Monitor test in the follow-up of patients with non-muscle invasive bladder cancer (NMIBC). METHODS: We conducted a prospective study on 1015 samples of 416 patients (mean age 72.2 ± 10.3 years) under follow-up for NMIBC. Patients underwent voided urinary cytology, the Xpert® Bladder Cancer Monitor test and cystoscopy and, if positive, a transurethral resection of the bladder. Xpert® Bladder Cancer Monitor was reported as negative or positive: cut-off total Linear Discriminant Analysis (LDA) = 0.5. RESULTS: We identified 168 recurrent tumours: 126 (75%) were low-grade (LG) and 42 (25%) high-grade (HG). Overall sensitivity was 17.9% for cytology, 52.4% for Xpert® Bladder Cancer Monitor and 54.2% for the two tests combined. The sensitivity of cytology increased from 6.3% in LG to 52.4% in HG tumours whereas Xpert® Bladder Cancer Monitor showed a sensitivity ranging from 42.9% in LG to 80.9% in HG tumours. Combined cytology and Xpert® Bladder Cancer Monitor yielded an overall sensitivity of 45.2% for LG and 80.9% for HG tumours. Overall specificity was 98.5% for cytology and 78.4% for Xpert® Bladder Cancer Monitor and 78.2% for the two tests combined. The area under the curve (AUC) for Xpert® Bladder Cancer Monitor was 0.71; stratifying the patients according to the European Association of Urology risk groups, the AUC was 0.69, 0.67 and 0.85 for low, intermediate and high risk, respectively (p = 0.0003). CONCLUSION: Our data confirm a significantly higher sensitivity of Xpert® Bladder Cancer Monitor than for cytology in a larger patient cohort. The test performed very well in terms of specificity but could not reach the high value of cytology. Along with voided urinary cytology the test could allow to reduce cystoscopies in follow-up patients, reducing discomfort to the patients and costs.

Comparison of 2 new real-time polymerase chain reaction-based urinary markers in the follow-up of patients with non-muscle-invasive bladder cancer.

BACKGROUND: The objective of the current study was to compare the diagnostic accuracy of 2 new real-time polymerase chain reaction-based urinary markers with each other and with urinary cytology, cystoscopy, and/or histology in patients being followed for non-muscle-invasive bladder cancer. METHODS: A total of 487 patients were enrolled in the study. Patients were evaluated using voided urine cytology, the Xpert Bladder Cancer Monitor, the Bladder EpiCheck test, and white light cystoscopy. RESULTS: The overall sensitivity was 27.17% for cytology, 64.13% for the Bladder EpiCheck test, and 66.3% for the Xpert Bladder Cancer Monitor. The overall specificity was 98.82% for cytology, 82.06% for the Bladder EpiCheck test, and 76.47% for the Xpert Bladder Cancer Monitor. The negative predictive value was very similar for the 3 tests at 83.56% for cytology, 89.42% for the Bladder EpiCheck test, and 89.35% for the Xpert Bladder Cancer Monitor. When combined, the Bladder EpiCheck test and Xpert Bladder Cancer Monitor detected overall 79.35% of the tumors: 70.37% in low-grade and 92.11% in high-grade tumors. CONCLUSIONS: The Xpert Bladder Cancer Monitor and Bladder EpiCheck test were found to perform very well in terms of sensitivity. Together, the 2 tests detected approximately 92.11% of high-grade tumors. Their specificity was high but could not reach the excellent value of cytology. The negative predictive value was the same for both tests and was higher than that for cytology, especially when the tests were used together (92.24%). These 2 new tests hold promise as urinary biomarkers. They may be used in combination to maximize sensitivity in a less invasive way, thereby reducing invasiveness in the follow-up of patients with non-muscle-invasive bladder cancer and decreasing discomfort for the patients as well as complications and costs.

Diagnostic predictive value of the Bladder EpiCheck test in the follow-up of patients with non-muscle-invasive bladder cancer.

BACKGROUND: The objective of this study was to evaluate the diagnostic accuracy of the Bladder EpiCheck test in the follow-up of patients with non-muscle-invasive bladder cancer (NMIBC) and to compare it with the accuracy of urinary cytology, cystoscopy, and/or histology. METHODS: In total, 243 patients were enrolled in the current study. Patients were evaluated by voided urine cytology, by the Bladder EpiCheck test, and by white-light cystoscopy. RESULTS: Overall sensitivity was 33.3% for cytology, 62.3% for Bladder EpiCheck, and 66.7% for the 2 tests combined. The sensitivity of cytology increased from 7.7% in low-grade (LG) tumors to 66.6% in high-grade (HG) tumors; whereas, for the Bladder EpiCheck test, the sensitivity was 46.1% in LG tumors and 83.3% in HG tumors. Combined cytology and Bladder EpiCheck testing yielded an overall sensitivity of 56.4% for LG tumors and 90% for HG tumors. Overall specificity was 98.6% for cytology, 86.3% for Bladder EpiCheck, and 85.6% for the 2 tests combined. The positive predictive value was 92% for cytology and 68.2% for Bladder EpiCheck. For the 2 tests combined, it was 68.6%. The negative predictive value was similar for the 2 tests: 75.8% for cytology, 82.9% for Bladder EpiCheck, and 84.5% for the 2 tests combined. CONCLUSIONS: The sensitivity of the Bladder EpiCheck test was significantly higher than that of cytology. The test performed very well in terms of specificity but could not reach the high value of cytology. The positive predictive value was higher for Bladder EpiCheck, whereas the negative predictive value was approximately the same for both tests.

Compound heterozygous SZT2 mutations in two siblings with early-onset epilepsy, intellectual disability and macrocephaly.

PURPOSE: Mutations in SZT2 have been previously reported in several cases of early onset epilepsy and intellectual disability. In this study we investigate potential causal mutations in two male siblings affected by early onset epilepsy, intellectual disability and macrocephaly. METHODS: We use family-based whole-exome sequencing to identify candidate variants. RESULTS: We report the identification of two potential causal SZT2 mutations in compound heterozygous state. We observe considerable differences in the clinical phenotype severity of the two affected individuals. The cerebral MRI revealed no abnormalities in the older affected brother, while in the youngest one it revealed a right frontal polymicrogiria. Moreover, while good seizure control was achieved in the older affected individual the younger brother is affected by pharmacoresistant epilepsy, progressive spastic paraplegia, cortical myoclonus and a more severe intellectual disability. We also analyzed the relative location of the reported pathogenic mutations in the SZT2 protein. CONCLUSION: Variable phenotypic expressivity is observed for this condition, while the location and type of mutations in SZT2 also has a potential impact on epilepsy severity. These findings extend our knowledge of epileptogenic conditions related to SZT2 and mTOR signaling.

Urachal carcinoma: from gross specimen to morphologic, immunohistochemical, and molecular analysis.

Urachal carcinoma (UrC) is an exceedingly rare neoplasm that develops from the urachus, an embryologic remnant of the urogenital sinus and allantois. The most commonly encountered histologic subtype is adenocarcinoma. The aim of this study is to characterize a series of UrC by morphology, immunohistochemistry, and molecular analysis. We retrospectively investigated seven cases of UrCs and assessed patient symptoms, imaging, histologic features, immunohistochemical profile, molecular characteristics, pathologic stages, and type of treatment. Immunostaining for CK7, CK20, Muc-2, CDX2, GATA3, ß-catenin, and CK34ßE12 was carried out on each neoplasm and on seven non-neoplastic urachal remnants as the control group. Additionally, a mutational analysis was performed using the QIAact Actionable Insights Tumor Panel Kit, which analyzes KRAS, NRAS, KIT, BRAF, PDGFRA, ALK, EGFR, ERBB2, PIK3CA, ERBB3, ESR1, and RAF1. Our cohort comprised five females and two males with a mean age of 64 years. UrCs consisted of two mucinous cystadenocarcinomas and five invasive, non-cystic adenocarcinomas. Carcinoma antigen expression profile was positive for CK20 and negative for CK34ßE12 and GATA3 in all cases. Five of seven cases stained positively for Muc-2 and CDX2. On the contrary, non-neoplastic urachal remnants were immunoreactive for CK34ßE12, CK7, and GATA3. Mutational analysis gave a positive result in four out of seven (57.1%) cases. All four positive tumors showed RAS mutation and one an additional mutation in PIK3CA. Urachal tumors exhibit peculiar morphologic, immunohistochemical, and molecular features. Due to the advanced stage at presentation, individualized treatment should be undertaken.

Diagnostic predictive value of Xpert Bladder Cancer Monitor in the follow-up of patients affected by non-muscle invasive bladder cancer.

AIMS: Cystoscopy and urine cytology represent the gold standard for monitoring superficial bladder cancer (BC). Xpert BC Monitor is a new urinary marker based on the evaluation of five target mRNAs overexpressed in patients with bladder cancer. The aim of our study was to evaluate the diagnostic accuracy of Xpert BC Monitor in follow-up of patients with non-muscle invasive bladder cancer (NMIBC). METHODS: 230 patients were included in this prospective study. Xpert BC Monitor cut-off was set to 0.5. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of cytology, Xpert BC Monitor and their combination were calculated and compared with cystoscopy/histology. RESULTS: 52/230 patients showed a NMIBC recurrence, 45 low grade (LG) and 7 high grade (HG). Overall sensitivity was 11.5% for cytology, 46.2% for Xpert BC Monitor and 48.1% for the two tests combined. Sensitivity of cytology increased from 4.4% in LG to 57.1% in HG tumours whereas for the Xpert BC Monitor it was 40% in LG and 85.7% in HG tumours. Combined cytology and Xpert BC Monitor yielded an overall sensitivity of 42% for LG and 85.7% for HG. Overall specificity was 97.2% for cytology, 77% for Xpert BC Monitor and 75.8% for the two tests. CONCLUSIONS: Sensitivity for the Xpert BC Monitor Test was significantly higher than for cytology. The test performed very well in terms of specificity but could not reach the value of cytology, while PPV and NPV performed approximately the same for both tests.

The PPARGC1A locus and CNS-specific PGC-1α isoforms are associated with Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17 kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17 kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (P = .0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.

Plasma and White Blood Cells Show Different miRNA Expression Profiles in Parkinson's Disease.

Parkinson's disease (PD) diagnosis is based on the assessment of motor symptoms, which manifest when more than 50% of dopaminergic neurons are degenerated. To date, no validated biomarkers are available for the diagnosis of PD. The aims of the present study are to evaluate whether plasma and white blood cells (WBCs) are interchangeable biomarker sources and to identify circulating plasma-based microRNA (miRNA) biomarkers for an early detection of PD. We profiled plasma miRNA levels in 99 L-dopa-treated PD patients from two independent data collections, in ten drug-naïve PD patients, and in unaffected controls matched by sex and age. We evaluated expression levels by reverse transcription and quantitative real-time PCR (RT-qPCR) and combined the results from treated PD patients using a fixed effect inverse-variance weighted meta-analysis. We revealed different expression profiles comparing plasma and WBCs and drug-naïve and L-dopa-treated PD patients. We observed an upregulation trend for miR-30a-5p in L-dopa-treated PD patients and investigated candidate target genes by integrated in silico analyses. We could not analyse miR-29b-3p, normally expressed in WBCs, due to the very low expression in plasma. We observed different expression profiles in WBCs and plasma, suggesting that they are both suitable but not interchangeable peripheral sources for biomarkers. We revealed miR-30a-5p as a potential biomarker for PD in plasma. In silico analyses suggest that miR-30a-5p might have a regulatory role in mitochondrial dynamics and autophagy. Further investigations are needed to confirm miR-30a-5p deregulation and targets and to investigate the influence of L-dopa treatment on miRNA expression levels.

Sequential recruitment of study participants may inflate genetic heritability estimates.

After the success of genome-wide association studies to uncover complex trait loci, attempts to explain the remaining genetic heritability (h 2) are mainly focused on unraveling rare variant associations and gene-gene or gene-environment interactions. Little attention is paid to the possibility that h 2 estimates are inflated as a consequence of the epidemiological study design. We studied the time series of 54 biochemical traits in 4373 individuals from the Cooperative Health Research In South Tyrol (CHRIS) study, a pedigree-based study enrolling ten participants/day over several years, with close relatives preferentially invited within the same day. We observed distributional changes of measured traits over time. We hypothesized that the combination of such changes with the pedigree structure might generate a shared-environment component with consequent h 2 inflation. We performed variance components (VC) h 2 estimation for all traits after accounting for the enrollment period in a linear mixed model (two-stage approach). Accounting for the enrollment period caused a median h 2 reduction of 4%. For 9 traits, the reduction was of >20%. Results were confirmed by a Bayesian Markov chain Monte Carlo analysis with all VCs included at the same time (one-stage approach). The electrolytes were the traits most affected by the enrollment period. The h 2 inflation was independent of the h 2 magnitude, laboratory protocol changes, and length of the enrollment period. The enrollment process may induce shared-environment effects even under very stringent and standardized operating procedures, causing h 2 inflation. Including the day of participation as a random effect is a sensitive way to avoid overestimation.

SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

The Cooperative Health Research in South Tyrol (CHRIS) study: rationale, objectives, and preliminary results.

The Cooperative Health Research In South Tyrol (CHRIS) study is a population-based study with a longitudinal lookout to investigate the genetic and molecular basis of age-related common chronic conditions and their interaction with life style and environment in the general population. All adults of the middle and upper Vinschgau/Val Venosta are invited, while 10,000 participants are anticipated by mid-2017. Family participation is encouraged for complete pedigree reconstruction and disease inheritance mapping. After a pilot study on the compliance with a paperless assessment mode, computer-assisted interviews have been implemented to screen for conditions of the cardiovascular, endocrine, metabolic, genitourinary, nervous, behavioral, and cognitive system. Fat intake, cardiac health, and tremor are assessed instrumentally. Nutrient intake, physical activity, and life-course smoking are measured semi-quantitatively. Participants are phenotyped for 73 blood and urine parameters and 60 aliquots per participant are biobanked (cryo-preserved urine, DNA, and whole and fractionated blood). Through liquid-chromatography mass-spectrometry analysis, metabolite profiling of the mitochondrial function is assessed. Samples are genotyped on 1 million variants with the Illumina HumanOmniExpressExome array and the first data release including 4570 fully phenotyped and genotyped samples is now available for analysis. Participants' follow-up is foreseen 6 years after the first visit. The target population is characterized by long-term social stability and homogeneous environment which should both favor the identification of enriched genetic variants. The CHRIS cohort is a valuable resource to assess the contribution of genomics, metabolomics, and environmental factors to human health and disease. It is awaited that this will result in the identification of novel molecular targets for disease prevention and treatment.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.