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Supplementary Methods, Tables 1-2 and Figures from Tumor Stroma–Derived Wnt5a Induces Differentiation of Basal Cell Carcinoma of Ptch-Mutant Mice via CaMKII

Colorectal cancer (CRC) incidence in young adults is rising. Identifying genetic risk factors is fundamental for the clinical management of patients and their families. This study aimed to identify clinically significant germline variants among young adults with CRC. Whole-exome sequencing data of blood-derived DNA from 133 unrelated young CRC patients (

Ubiquitous overactivation of Hedgehog signaling in adult pituitaries results in increased expression of proopiomelanocortin (Pomc), growth hormone (Gh) and prolactin (Prl), elevated adrenocorticotropic hormone (Acth) production and proliferation of Sox2+ cells. Moreover, ACTH, GH and PRL-expressing human pituitary adenomas strongly express the Hedgehog target GLI1. Accordingly, Hedgehog signaling seems to play an important role in pathology and probably also in homeostasis of the adult hypophysis. However, the specific Hedgehog-responsive pituitary cell type has not yet been identified. We here investigated the Hedgehog pathway activation status and the effects of deregulated Hedgehog signaling cell-specifically in endocrine and non-endocrine pituitary cells. We demonstrate that Hedgehog signaling is unimportant for the homeostasis of corticotrophs, whereas it is active in subpopulations of somatotrophs and folliculo-stellate cells in vivo. Reinforcement of Hedgehog signaling activity in folliculo-stellate cells stimulates growth hormone production/release from somatotrophs in a paracrine manner, which most likely is mediated by the neuropeptide vasoactive intestinal peptide. Overall, our data show that Hedgehog signaling affects the homeostasis of pituitary hormone production via folliculo-stellate cell-mediated regulation of growth hormone production/secretion.

Aminoacylation of tRNA is a key step in protein biosynthesis, carried out by highly specific aminoacyl-tRNA synthetases (ARS). ARS have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl-tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl-tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N-terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1-related syndrome and define an emerging disease spectrum: aminoacyl-tRNA synthetase-related developmental disorders with or without microcephaly.

BACKGROUND Hereditary angioedema (HAE) is a group of genetic diseases characterized by recurrent, painful and potentially lethal tissue swelling. The most common form results from mutations in the SERPING1 gene, leading to reduced function of complement 1 inhibitor (C1-INH). Rarer forms with normal C1-INH may arise from mutations in the coagulation factor F12 gene, but mostly the genetic background is unknown. Recently, a novel HAE mutation in the plasminogen (PLG) gene was shown. PATIENTS AND METHODS We analyzed the various clinical manifestations of HAE in 14 related patients using clinical data, biochemical analysis for C1-INH and C4 as well as gene sequencing. RESULTS Patients' symptoms were assigned to two different forms of HAE. In ten patients suffering from swelling of the lips or tongue but not of the extremities, a mutation in the PLG gene (c.988A>G) was found whereas in the only four patients with swelling of the gastrointestinal tract and extremities, a mutation in the SERPING1 gene (c.1480C>T) was identified. In two cases this was additional to PLG c.988A>G. CONCLUSIONS This unique finding of two different HAE-specific mutations in a large family not only explains the divergent phenotypes but also supports a genotype-phenotype correlation showing that abdominal attacks and swelling of the extremities are common with HAE-C1-INH but unusual with HAE-PLG.

Bloom syndrome (BS) is an autosomal recessive disease clinically characterized by primary microcephaly, growth deficiency, immunodeficiency, and predisposition to cancer. It is mainly caused by biallelic loss-of-function mutations in the BLM gene, which encodes the BLM helicase, acting in DNA replication and repair processes. Here, we describe the gene expression profiles of three BS fibroblast cell lines harboring causative, biallelic truncating mutations obtained by single-cell (sc) transcriptome analysis. We compared the scRNA transcription profiles from three BS patient cell lines to two age-matched wild-type controls and observed specific deregulation of gene sets related to the molecular processes characteristically affected in BS, such as mitosis, chromosome segregation, cell cycle regulation, and genomic instability. We also found specific upregulation of genes of the Fanconi anemia pathway, in particular FANCM, FANCD2, and FANCI, which encode known interaction partners of BLM. The significant deregulation of genes associated with inherited forms of primary microcephaly observed in our study might explain in part the molecular pathogenesis of microcephaly in BS, one of the main clinical characteristics in patients. Finally, our data provide first evidence of a novel link between BLM dysfunction and transcriptional changes in condensin complex I and II genes. Overall, our study provides novel insights into gene expression profiles in BS on a single-cell level, linking specific genes and pathways to BLM dysfunction.

The role of RNF43 as a cause of an inherited predisposition to colorectal cancer (CRC) is yet to be fully explored. This report presents our findings of two individuals with CRC from a single family carrying a likely-pathogenic inherited germline variant in RNF43. The proband (III:1) and the proband's mother (II:2) were diagnosed with mismatch repair proficient CRCs at the age of 50 years and 65 years, respectively. Both patients had BRAFⱽ⁶⁰⁰ᴱ mutated colon tumours, indicating that the CRCs arose in sessile serrated lesions. The germline variant RNF43:c.375+1G>A was identified in both patients. RNA studies showed that this variant resulted in an aberrantly spliced transcript, which was predicted to encode RNF43:p.Ala126Ilefs*50 resulting in premature termination of protein synthesis and was classified as a likely-pathogenic variant. Our report adds further evidence to the hereditary role of RNF43 as a tumour suppressor gene in colorectal tumorigenesis and supports the inclusion of RNF43 as a gene of interest in the investigation of CRC predispositions outside the setting of serrated polyposis. Refereed/Peer-reviewed

Background/Purpose Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and contractile dysfunction. Fibrosis is one major phenotypic result in DCM, pointing to the contribution of both, cardiomyocytes (CM) and cardiac fibroblasts (cFB) to DCM. The molecular basis of most DCM cases remains unknown. Nevertheless, it is known that up to 35% of all cases have a family history, linked to mutations in more than 30 gene loci. The aim of this study is to analyse the crosstalk of iPSC-CM and cFB and the underlying genetic and molecular causes in a patient-specific induced pluripotent stem cell (iPSC) model of DCM. Methods and results For this purpose a 4-member family was recruited containing 2 patients (father and daughter) with severe DCM and heart transplantation. iPSCs of all family members were generated and differentiated into iPSC-CMs. All iPSC-CMs express general cardiac markers, e.g. βMHC, α-actinin. Interestingly, αMHC expression was decreased in diseased iPSC-CMs in comparison to control cells. Additionally, the sarcomeric regularity was decreased in diseased iPSC-CMs. As we found significantly increased fibrosis (22%) in explanted myocardium of the diseased father compared to healthy myocardium (8%), both cFB and CM seem to play an important role. From the same myocardium primary cFBs were isolated and shown to express typical cFB markers clearly distinguishing these cells from non-fibroblasts as well as from fibroblasts with different origin. To analyse the contribution of cFBs and CMs to DCM on a functional level, 3D engineered heart muscles (EHMs) were generated in different diseased/healthy cell combinations. EHMs composed of both or either one affected DCM-iPSC-CMs and/or DCM-cFBs in comparison to healthy control EHMs did not produce any measurable force, indicating that the DCM-EHM phenotype is clearly diseased. Evaluation of tissues' viscoelasticity showed that DCM-cFB, DCM-iPSC-CMs and DCM-EHMs were stiffer than healthy control EHMs. Thus these data suggest that apart from the obvious dysfunction of DCM CMs, DCM cFBs clearly contribute to the contractile pathophysiology in DCM EHMs. Furthermore, whole exome sequencing of iPSCs was conducted to identify disease-causing variants. This analyses point towards a new genetic variant in the FLNc gene coding for a protein important in development, stabilization and maintenance of myofibrils. Rescue of this variant by CRISPR Cas9 genome editing will shed more light onto the role of this variant during DCM development in the future. Conclusion Using a ps-iPSC-CM model of a 4-member family with two severe DCM patients, we could demonstrate a clear contribution of both cell types, iPSC-CMs and cFB, to the contractile pathophysiology of DCM. We identified a potentially disease-causing new variant in the FLNc gene, which may contribute to the impaired functionality within the diseased iPSC-CMs and EHM. This makes FLNc a new therapeutic target for DCM. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): IRTG1816

Dilated cardiomyopathy (DCM) belongs to the most frequent forms of cardiomyopathy mainly characterized by cardiac dilatation and reduced systolic function. Although most cases of DCM are classified as sporadic, 20–30% of cases show a heritable pattern. Familial forms of DCM are genetically heterogeneous, and mutations in several genes have been identified that most commonly play a role in cytoskeleton and sarcomere-associated processes. Still, a large number of familial cases remain unsolved. Here, we report five individuals from three independent families who presented with severe dilated cardiomyopathy during the neonatal period. Using whole-exome sequencing (WES), we identified causative, compound heterozygous missense variants in RPL3L (ribosomal protein L3-like) in all the affected individuals. The identified variants co-segregated with the disease in each of the three families and were absent or very rare in the human population, in line with an autosomal recessive inheritance pattern. They are located within the conserved RPL3 domain of the protein and were classified as deleterious by several in silico prediction software applications. RPL3L is one of the four non-canonical riboprotein genes and it encodes the 60S ribosomal protein L3-like protein that is highly expressed only in cardiac and skeletal muscle. Three-dimensional homology modeling and in silico analysis of the affected residues in RPL3L indicate that the identified changes specifically alter the interaction of RPL3L with the RNA components of the 60S ribosomal subunit and thus destabilize its binding to the 60S subunit. In conclusion, we report that bi-allelic pathogenic variants in RPL3L are causative of an early-onset, severe neonatal form of dilated cardiomyopathy, and we show for the first time that cytoplasmic ribosomal proteins are involved in the pathogenesis of non-syndromic cardiomyopathies.

Nine years of sampling and analyses of fine particles (PM2.5) were performed in Beijing from 2005 to 2013. Twenty-seven chemical elements and black carbon (BC) in PM2.5 were analyzed in order to study chemical characteristics and temporal distribution of Beijing aerosols. Principle component analysis defined different types of elemental sources, based on which, the influences of a variety of anthropogenic activities including governmental intervention measures and natural sources on air quality were evaluated. For the first time, Ga is used as a tracer element for heating activities mainly using coal in Beijing, due to its correlation with BC and coal combustion, as well as its concentration variation between the heating- and non-heating periods. The traffic restrictions effectively reduced emissions of relevant heavy metals such as As, Cd, Sn and Sb. The expected long-term effectiveness of the steel smelters relocation was not observed due to the nearby relocation with increased capacity. Firework display during every Chinese spring festival season and special events such as the Olympic Games resulted in several times higher concentrations of K, Sr and Ba than other days and thus they were proposed as tracers for firework display. The impacts of all these factors were quantified and evaluated. Sand dust or dust storms induced higher concentrations of geogenic elements in PM2.5 compared to non-dust days. Sustainable mitigation measures, such as traffic restrictions, are necessary to be continued and improved to obtain more "blue sky" days in the future.

We investigated a four-generation family of German ancestry with distal myopathy. Four individuals in two generations were affected. Foot and toe extensor paresis progressing very slowly over decades was the core neurological sign, reflected by fatty infiltration of the lower leg extensor muscles on muscle MRI. Additionally, finger extensor paresis was present in two patients and quadriceps muscle paresis in one. Distal sensory signs had initially given rise to the diagnosis of axonal Charcot-Marie-Tooth (CMT) disease. Two patients had extended verrucae of their foot sole, which may or may not be part of the disease spectrum. All four patients had a novel c.4645G > C mutation in exon 34 of the MYH7 gene that was not present in three clinically unaffected family members. Muscle biopsy of one patient revealed a myopathic pattern associated with type 1 muscle fibre atrophy and core-like lesions in many muscle fibres consistent with a myosin-related myopathy. We conclude that some of the typical clinical signs such as extensor weakness of the big toe and the little finger may only develop in the further course of the disease.

Incorporating tailored nanostructures into solar cells is a promising way to improve their photovoltaic conversion efficiency. For such solar cells, sputtered indium-doped tin oxide on glass is the standard substrate. As the global resources of indium are very limited, there is the challenge to move to other substrates and thus to synthesize nanostructures on these. We report on the growth and characterization of zinc oxide nanorod arrays on indium-free transparent conducting oxide substrates, in particular on sputtered aluminum-doped ZnO. We present a catalyst-free vapor-transport growth method at 510 °C, which allows for the growth of highly crystalline, well-aligned ZnO nanorods arrays with a tunable length, ranging from hundreds of nanometers to several micrometers. The nanorods exhibit an excellent crystal quality, as shown by photoluminescence measurements and high-resolution transmission electron microscopy. Optical transmission spectra show a sufficient transparency of the substrates, qualifying t...

Background: Mutations in the SPG4/SPAST gene are the most common cause for hereditary spastic paraplegia (HSP). The splice-site mutations make a significant contribution to HSP and account for 17.4% of all types of mutations and 30.8% of point mutations in the SPAST gene. However, only few studies with limited molecular approach were conducted to investigate and decipher the role of SPAST splice-site mutations in HSP. Methods: A reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative allele-specific expression assay were performed. Results: We have characterized the consequence of two novel splice-site mutations (c.1493 + 1G>A and c.1414−1G>A) in the SPAST gene in two different families with pure HSP. The RT-PCR analysis revealed that both spastin mutations are indeed splice-site mutations and cause skipping of exon 12. Furthermore, RT-PCR data suggested that these splice-site mutations may cause leaky splicing. By means of a quantitative allele-specific expression assay, we could confirm that both splice-site mutations cause leaky splicing, as the relative expression of the exon 12-skipped transcript was reduced (21.1 ± 3.6 compared to expected 50%). Conclusions: Our finding supports a “threshold-effect-model” for functional spastin in HSP. A higher level (78.8 ± 3.9%) of functional spastin than the expected ratio of 50% owing to leaky splicing might cause late age at onset of HSP. Remarkably, we could show that a quantitative allele-specific expression assay is a simple and effective tool to evaluate the role of most types of spastin splice-site mutations in HSP.