Rare Variants in BNC2 Are Implicated in Autosomal-Dominant Congenital Lower Urinary-Tract Obstruction.

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.

BRAFV600E and BRAF-WT Specific Antitumor Immunity in Papillary Thyroid Cancer.

One feature of papillary thyroid cancer (PTC) is the frequently present somatic BRAFV600E mutation. PTCs are also characterized by a lymphocytic infiltration, which may correlate with an improved clinical outcome. The objective of the study was the characterization of BRAFV600E specific anti-immunity in PTC patients and correlation analyses with the clinical outcome. Fourteen HLA A2 positive PTC patients were included into the study of whom tumor tissue samples were also available. Of those, 8 PTC patients revealed a somatic BRAFV600E mutation. All PTC patients were also MHC class II typed. Tetramer analyses for detection of MHC class I and MHC class II-restricted, BRAFV600E epitope-specific T cells using unstimulated and peptide-stimulated T cells were performed; correlation analyses between MHC phenotypes, T cell immunity, and the clinical course were performed. In regard to unstimulated T cells, a significantly higher amount of BRAFV600E epitope specific T cells was detected compared to a control tetramer. Importantly, after overnight peptide stimulation a significantly higher number of BRAFV600E positive and BRAF WT epitope-specific T cells could be seen. In regard to the clinical course, however, no significant differences were seen, neither in the context of the initial tumor size, nor in the context of lymph node metastases or peripheral metastastic spread. In conclusion, we clearly demonstrated a BRAF-specific tumor immunity in PTC-patients which is, however, independent of a BRAFV600E status of the PTC patients.

Recognizable Pattern of Arthrogryposis and Congenital Myopathy Caused by the Recurrent TTN Metatranscript-only c.39974-11T > G Splice Variant.

INTRODUCTION: Arthrogryposis is characterized by the presence of multiple contractures at birth and can be caused by pathogenic variants in TTN (Titin). Exons and variants that are not expressed in one of the three major isoforms of titin are referred to as "metatranscript-only" and have been considered to be only expressed during fetal development. Recently, the metatranscript-only variant (c.39974-11T > G) in TTN with a second truncating TTN variant has been linked to arthrogryposis multiplex congenita and myopathy. METHODS: Via exome sequencing we identified the TTN c.39974-11T > G splice variant in trans with one of three truncating variants (p.Arg8922*, p.Lys32998Asnfs*63, p.Tyr10345*) in five individuals from three families. Clinical presentation and muscle ultrasound as well as MRI images were analyzed. RESULTS: All five patients presented with generalized muscular hypotonia, reduced muscle bulk, and congenital contractures most prominently affecting the upper limbs and distal joints. Muscular hypotonia persisted and contractures improved over time. One individual, the recipient twin in the setting of twin-to-twin transfusion syndrome, died from severe cardiac hypertrophy 1 day after birth. Ultrasound and MRI imaging studies revealed a recognizable pattern of muscle involvement with striking fibrofatty involvement of the hamstrings and calves, and relative sparing of the femoral adductors and anterior segment of the thighs. CONCLUSION: The recurrent TTN c.39974-11T > G variant consistently causes congenital arthrogryposis and persisting myopathy providing evidence that the metatranscript-only 213 to 217 exons impact muscle elasticity during early development and beyond. There is a recognizable pattern of muscle involvement, which is distinct from other myopathies and provides valuable clues for diagnostic work-up.

CNS myelin protein 36K regulates oligodendrocyte differentiation through Notch.

In contrast to humans and other mammals, zebrafish can successfully regenerate and remyelinate central nervous system (CNS) axons following injury. In addition to common myelin proteins found in mammalian myelin, 36K protein is a major component of teleost fish CNS myelin. Although 36K is one of the most abundant proteins in zebrafish brain, its function remains unknown. Here we investigate the function of 36K using translation-blocking Morpholinos. Morphant larvae showed fewer dorsally migrated oligodendrocyte precursor cells as well as upregulation of Notch ligand. A gamma secretase inhibitor, which prevents activation of Notch, could rescue oligodendrocyte precursor cell numbers in 36K morphants, suggesting that 36K regulates initial myelination through inhibition of Notch signaling. Since 36K like other short chain dehydrogenases might act on lipids, we performed thin layer chromatography and mass spectrometry of lipids and found changes in lipid composition in 36K morphant larvae. Altogether, we suggest that during early development 36K regulates membrane lipid composition, thereby altering the amount of transmembrane Notch ligands and the efficiency of intramembrane gamma secretase processing of Notch and thereby influencing oligodendrocyte precursor cell differentiation and further myelination. Further studies on the role of 36K short chain dehydrogenase in oligodendrocyte precursor cell differentiation during remyelination might open up new strategies for remyelination therapies in human patients.

Isomorphic diffuse glioma is a morphologically and molecularly distinct tumour entity with recurrent gene fusions of MYBL1 or MYB and a benign disease course.

The "isomorphic subtype of diffuse astrocytoma" was identified histologically in 2004 as a supratentorial, highly differentiated glioma with low cellularity, low proliferation and focal diffuse brain infiltration. Patients typically had seizures since childhood and all were operated on as adults. To define the position of these lesions among brain tumours, we histologically, molecularly and clinically analysed 26 histologically prototypical isomorphic diffuse gliomas. Immunohistochemically, they were GFAP-positive, MAP2-, OLIG2- and CD34-negative, nuclear ATRX-expression was retained and proliferation was low. All 24 cases sequenced were IDH-wildtype. In cluster analyses of DNA methylation data, isomorphic diffuse gliomas formed a group clearly distinct from other glial/glio-neuronal brain tumours and normal hemispheric tissue, most closely related to paediatric MYB/MYBL1-altered diffuse astrocytomas and angiocentric gliomas. Half of the isomorphic diffuse gliomas had copy number alterations of MYBL1 or MYB (13/25, 52%). Gene fusions of MYBL1 or MYB with various gene partners were identified in 11/22 (50%) and were associated with an increased RNA-expression of the respective MYB-family gene. Integrating copy number alterations and available RNA sequencing data, 20/26 (77%) of isomorphic diffuse gliomas demonstrated MYBL1 (54%) or MYB (23%) alterations. Clinically, 89% of patients were seizure-free after surgery and all had a good outcome. In summary, we here define a distinct benign tumour class belonging to the family of MYB/MYBL1-altered gliomas. Isomorphic diffuse glioma occurs both in children and adults, has a concise morphology, frequent MYBL1 and MYB alterations and a specific DNA methylation profile. As an exclusively histological diagnosis may be very challenging and as paediatric MYB/MYBL1-altered diffuse astrocytomas may have the same gene fusions, we consider DNA methylation profiling very helpful for their identification.

Recent aspects of classification and epidemiology of epilepsy-associated tumors.

Epileptic seizures are frequent manifestations of brain tumors. However, biopsy specimens of patients who undergo neurosurgical removal of circumscribed foci to control chronic recurrent pharmacoresistant seizures often reveal tumor entities that are rare in general brain tumor series. The spectrum of these "long-term epilepsy-associated neoplasms" comprises highly differentiated glial and glioneuronal tumors that show a benign biologic behavior and clinical course, and that rarely relapse. Several entities are well recognizable on the basis of histopathologic and immunohistochemical characteristics. An intriguing functional aspect of these tumors, sometimes collectively referred to as "epileptomas," is their prominent epileptogenicity, which may represent a clinical feature indicating rather than causing the generally benign biologic behavior of these tumors. A frequent feature of respective neoplasms is their coincidence with dysplastic lesions in the vicinity of the tumor itself. The recent advent of new molecular markers, including genomic alterations leading to activation of the protooncogene BRAF and impaired function of isocitrate dehydrogenase (IDH1), provides excellent new tools in the differential diagnosis of low grade brain tumors, and provides intriguing implications to further develop the pathogenetic concepts of these neoplasms. Despite this progress, a number of tumors from patients with chronic epilepsy show combinations of cytologic, histologic, and immunohistochemical characteristics that challenge the current neuropathologic classification schemes. Attempts are currently ongoing to develop further classification schemes.

Whole Exome Analysis to Select Targeted Therapies for Patients with Metastatic Breast Cancer - A Feasibility Study.

Introduction: The purpose of this feasibility study was to select targeted therapies according to "ESMO Scale for Clinical Actionability of molecular Targets (ESCAT)". Data interpretation was further supported by a browser-based Treatment Decision Support platform (MH Guide, Molecular Health, Heidelberg, Germany). Patients: We applied next generation sequencing based whole exome sequencing of tumor tissue and peripheral blood of patients with metastatic breast cancer (n = 44) to detect somatic as well as germline mutations. Results: In 32 metastatic breast cancer patients, data interpretation was feasible. We identified 25 genomic alterations with ESCAT Level of Evidence I or II in 18/32 metastatic breast cancer patients, which were available for evaluation: three copy number gains in HER2 , two g BRCA1 , two g BRCA2 , six PIK3CA, one ESR1 , three PTEN , one AKT1 and two HER2 mutations. In addition, five samples displayed Microsatellite instability high-H. Conclusions: Resulting treatment options were discussed in a tumor board and could be recommended in a small but relevant proportion of patients with metastatic breast cancer (7/18). Thus, this study is a valuable preliminary work for the establishment of a molecular tumor board within the German initiative "Center for Personalized Medicine" which aims to shorten time for analyses and optimize selection of targeted therapies.

Humanized zebrafish as a tractable tool for in vivo evaluation of pro-myelinating drugs.

Therapies that promote neuroprotection and axonal survival by enhancing myelin regeneration are an unmet need to prevent disability progression in multiple sclerosis. Numerous potentially beneficial compounds have originated from phenotypic screenings but failed in clinical trials. It is apparent that current cell- and animal-based disease models are poor predictors of positive treatment options, arguing for novel experimental approaches. Here we explore the experimental power of humanized zebrafish to foster the identification of pro-remyelination compounds via specific inhibition of GPR17. Using biochemical and imaging techniques, we visualize the expression of zebrafish (zf)-gpr17 during the distinct stages of oligodendrocyte development, thereby demonstrating species-conserved expression between zebrafish and mammals. We also demonstrate species-conserved function of zf-Gpr17 using genetic loss-of-function and rescue techniques. Finally, using GPR17-humanized zebrafish, we provide proof of principle for in vivo analysis of compounds acting via targeted inhibition of human GPR17. We anticipate that GPR17-humanized zebrafish will markedly improve the search for effective pro-myelinating pharmacotherapies.

SLC20A1 Is Involved in Urinary Tract and Urorectal Development.

Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.

OTX2 Defines a Subgroup of Atypical Teratoid Rhabdoid Tumors With Close Relationship to Choroid Plexus Tumors.

Atypical teratoid rhabdoid tumors (ATRT) are highly malignant brain tumors of early childhood that have been regarded as a homogenous entity characterized by inactivation of the SMARCB1/INI1 or SMARCA4/BRG1 genes as the only characteristic alteration. Recent studies suggest that similar to other embryonal tumors ATRT can also be divided into subgroups based on their mRNA or methylation profiles. Using microarray-based expression analysis of 12 patient ATRT specimens we demonstrated the existence of 2 subgroups of ATRT. One subgroup is characterized by high expression of OTX2, encoding a transcription factor involved in brain development. OTX2 expression was verified by immunohistochemistry and might function as a novel therapeutic target for this fatal tumor. High expression of OTX2 as well as expression of Kir7.1/KCNJ13, TRPM3 and ENPP2, which have all previously been linked to either choroid plexus epithelium or choroid plexus tumors (CPTs), suggests a close histogenetic relation of this subgroup to CPTs.

High-Resolution Genomic Analysis of Cribriform Neuroepithelial Tumors of the Central Nervous System.

Cribriform neuroepithelial tumors (CRINET) are one of several recently characterized entities in the broad spectrum of solid tumors with SMARCB1-INI1 loss. This neoplasm seems to be exceedingly rare and displays unique neuropathologic and clinical features. To date, only a few cases of CRINET have been characterized from a molecular point of view. In this study, we investigated the molecular features of 3 cases of CRINET using multiplex ligation-dependent probe amplification and molecular inversion profiling approaches. Along with mutations and deletions of SMARCB1-INI1, molecular inversion profiling analysis revealed a stable genomic profile without significant large chromosomal changes. Focal alterations (gains) were observed in individual cases at chromosomes 4q12 (PDGFRA), 12q15 (MDM2), 7p15.1 (NPY), and 18q11.2 (CDH2). Genomic Identification of Significant Targets in Cancer analysis highlighted focal alterations, including gains at chromosomes 16q23.2 (MAF), 17q23 (AXIN2), and 8p12 (ADAM3A). No cases showed BRAF(V600E) or CTNNB1 mutations. These data indicate that CRINET present stable genetic features and lack alterations commonly identified in other pediatric brain tumors. Further studies are required to determine whether specific alterations and specific signaling pathways, in addition to SMARCB1-INI1, may be implicated in the biology of this rare tumor and whether there are additional molecular similarities between CRINET and atypical teratoid/rhabdoid tumors.

Molecular heterogeneity characterizes glioblastoma with lipoblast/adipocyte-like cytology.

Glioblastoma, the most common primary brain tumor in adults, may rarely show among unusual histological patterns lipoblast/adipocyte-like features. The genetic features of such cases are not yet well characterized, and molecular data are available for only few cases. In order to further expand our knowledge about their molecular profile, we analyzed four cases of glioblastoma with adipocyte-like features. Multiplex ligation-dependent probe amplification (MLPA) revealed loss of PTEN and MDM2 amplification in two cases while another case was characterized by CDKN2A deletion. Conversely, we did not find any evidence of EGFR amplification, BRAF (V600E) or IDH1/2 mutations. Our results, along with data published in previous studies, showed that glioblastoma with lipoblast/adipocyte-like cytology present a heterogeneous genetic background and therefore seem to represent more a rare phenotypic variant than a specific tumor subtype.

High-resolution genomic analysis does not qualify atypical plexus papilloma as a separate entity among choroid plexus tumors.

Choroid plexus tumors are rare neoplasms that mainly affect children. They include papillomas, atypical papillomas, and carcinomas. Detailed genetic studies are rare, and information about their molecular pathogenesis is limited. Molecular inversion probe analysis is a hybridization-based method that represents a reliable tool for the analysis of highly fragmented formalin-fixed paraffin-embedded tissue-derived DNA. Here, analysis of 62 cases showed frequent hyperdiploidy in papillomas and atypical papillomas that appeared very similar in their cytogenetic profiles. In contrast, carcinomas showed mainly losses of chromosomes. Besides recurrent focal chromosomal gains common to all choroid plexus tumors, including chromosome 14q21-q22 (harboring OTX2), chromosome 7q22 (LAMB1), and chromosome 9q21.12 (TRPM3), Genomic Identification of Significant Targets in Cancer analysis uncovered focal alterations specific for papillomas and atypical papillomas (e.g. 7p21.3 [ARL4A]) and for carcinomas (16p13.3 [RBFOX1] and 6p21 [POLH, GTPBP2, RSPH9, and VEGFA]). Additional RNA expression profiling and gene set enrichment analysis revealed greater expression of cell cycle-related genes in atypical papillomas in comparison with that in papillomas. These findings suggest that atypical papillomas represent an immature variant of papillomas characterized by increased proliferative activity, whereas carcinomas seem to represent a genetically distinct tumor group.

Identification and characterization of OSTL (RNF217) encoding a RING-IBR-RING protein adjacent to a translocation breakpoint involving ETV6 in childhood ALL.

Genomic aberrations involving ETV6 on band 12p13 are amongst the most common chromosomal abnormalities in human leukemia. The translocation t(6;12)(q23;13) in a childhood B-cell acute lymphoblastic leukemia (ALL) cell line fuses ETV6 with the putative long non-coding RNA gene STL. Linking STL properties to leukemia has so far been difficult. Here, we describe a novel gene, OSTL (annotated as RNF217 in Genbank), which shares the first exon and a CpG island with STL but is transcribed in the opposite direction. Human RNF217 codes for a highly conserved RING finger protein and is mainly expressed in testis and skeletal muscle with different splice variants. RNF217 shows regulated splicing in B cell development, and is expressed in a number of human B cell leukemia cell lines, primary human chronic myeloid leukemia, acute myeloid leukemia with normal karyotype and acute T-ALL samples. Using a yeast two-hybrid screen, we identified the anti-apoptotic protein HAX1 to interact with RNF217. This interaction could be mapped to the C-terminal RING finger motif of RNF217. We propose that some of the recurring aberrations involving 6q might deregulate the expression of RNF217 and result in imbalanced apoptosis signalling via HAX1, promoting leukemia development.