Re-analysis of whole genome sequencing ends a diagnostic odyssey: Case report of an RNU4-2 related neurodevelopmental disorder.

Despite increasing knowledge of disease-causing genes in human genetics, approximately half of the individuals affected by neurodevelopmental disorders remain genetically undiagnosed. Part of this missing heritability might be caused by genetic variants outside of protein-coding genes, which are not routinely diagnostically investigated. A recent preprint identified de novo variants in the non-coding spliceosomal snRNA gene RNU4-2 as a cause of a frequent novel syndromic neurodevelopmental disorder. Here we mined 164 whole genome sequencing (WGS) trios from individuals with neurodevelopmental or multiple congenital anomaly disorders that received diagnostic genomic investigations at our clinic. We identify a recurrent de novo RNU4-2 variant (NR_003137.2(RNU4-2):n.64_65insT) in a 5-year-old girl with severe global developmental delay, hypotonia, microcephaly, and seizures that likely explains her phenotype, given that extensive previous genetic investigations failed to identify an alternative cause. We present detailed phenotyping of the individual obtained during a 5-year follow-up. This includes photographs showing recognizable facial features for this novel disorder, which might allow prioritizing other currently unexplained affected individuals sharing similar facial features for targeted investigations of RNU4-2. This case illustrates the power of re-analysis to solve previously unexplained cases even when a diagnostic genome remains negative.

deCLUTTER2+ - a pipeline to analyze calcium traces in a stem cell model for ventral midbrain patterned astrocytes.

Astrocytes are the most populous cell type of the human central nervous system and are essential for physiological brain function. Increasing evidence suggests multiple roles for astrocytes in Parkinson's disease, nudging a shift in the research focus, which historically pivoted around ventral midbrain dopaminergic neurons (vmDANs). Studying human astrocytes and other cell types in vivo remains challenging. However, in vitro-reprogrammed human stem cell-based models provide a promising alternative. Here, we describe a novel protocol for astrocyte differentiation from human stem cell-derived vmDAN-generating progenitors. This protocol simulates the regionalization, gliogenic switch, radial migration and final differentiation that occur in the developing human brain. We characterized the morphological, molecular and functional features of these ventral midbrain patterned astrocytes with a broad palette of techniques and identified novel candidate midbrain-astrocyte specific markers. In addition, we developed a new pipeline for calcium imaging data analysis called deCLUTTER2+ (deconvolution of Ca2+ fluorescent patterns) that can be used to discover spontaneous or cue-dependent patterns of Ca2+ transients. Altogether, our protocol enables the characterization of the functional properties of human ventral midbrain patterned astrocytes under physiological conditions and in disease.

PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability.

The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signalling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau and LRRK2. Here, we report the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. We carried out clinical studies and genetic analyses, including genome-wide linkage analysis, whole-exome sequencing, and Sanger sequencing of candidate variants. We next performed functional studies on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. We first identified a homozygous PTPA variant, c.893T>G (p.Met298Arg), in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C>A (p.Ala171Asp), in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA orthologue knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. We conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Our findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.

A multiplex pedigree with pathologically confirmed multiple system atrophy and Parkinson's disease with dementia.

Multiple system atrophy is considered a sporadic disease, but neuropathologically confirmed cases with a family history of parkinsonism have been occasionally described. Here we report a North-Bavarian (colloquially, Lion's tail region) six-generation pedigree, including neuropathologically confirmed multiple system atrophy and Parkinson's disease with dementia. Between 2012 and 2020, we examined all living and consenting family members of age and calculated the risk of prodromal Parkinson's disease in those without overt parkinsonism. The index case and one paternal cousin with Parkinson's disease with dementia died at follow-up and underwent neuropathological examination. Genetic analysis was performed in both and another family member with Parkinson's disease. The index case was a female patient with cerebellar variant multiple system atrophy and a positive maternal and paternal family history for Parkinson's disease and dementia in multiple generations. The families of the index case and her spouse were genealogically related, and one of the spouse's siblings met the criteria for possible prodromal Parkinson's disease. Neuropathological examination confirmed multiple system atrophy in the index case and advanced Lewy body disease, as well as tau pathology in her cousin. A comprehensive analysis of genes known to cause hereditary forms of parkinsonism or multiple system atrophy lookalikes was unremarkable in the index case and the other two affected family members. Here, we report an extensive European pedigree with multiple system atrophy and Parkinson`s disease suggesting a complex underlying α-synucleinopathy as confirmed on neuropathological examination. The exclusion of known genetic causes of parkinsonism or multiple system atrophy lookalikes suggests that variants in additional, still unknown genes, linked to α-synucleinopathy lesions underlie such neurodegenerative clustering.

Correcting Differential Gene Expression Analysis for Cyto-Architectural Alterations in Substantia Nigra of Parkinson's Disease Patients Reveals Known and Potential Novel Disease-Associated Genes and Pathways.

Several studies have analyzed gene expression profiles in the substantia nigra to better understand the pathological mechanisms causing Parkinson's disease (PD). However, the concordance between the identified gene signatures in these individual studies was generally low. This might have been caused by a change in cell type composition as loss of dopaminergic neurons in the substantia nigra pars compacta is a hallmark of PD. Through an extensive meta-analysis of nine previously published microarray studies, we demonstrated that a big proportion of the detected differentially expressed genes was indeed caused by cyto-architectural alterations due to the heterogeneity in the neurodegenerative stage and/or technical artefacts. After correcting for cell composition, we identified a common signature that deregulated the previously unreported ammonium transport, as well as known biological processes such as bioenergetic pathways, response to proteotoxic stress, and immune response. By integrating with protein interaction data, we shortlisted a set of key genes, such as LRRK2, PINK1, PRKN, and FBXO7, known to be related to PD, others with compelling evidence for their role in neurodegeneration, such as GSK3ß, WWOX, and VPC, and novel potential players in the PD pathogenesis. Together, these data show the importance of accounting for cyto-architecture in these analyses and highlight the contribution of multiple cell types and novel processes to PD pathology, providing potential new targets for drug development.

A new alpha-synuclein missense variant (Thr72Met) in two Turkish families with Parkinson's disease.

INTRODUCTION: Missense variants and multiplications of the alpha-synuclein gene (SNCA) are established as rare causes of autosomal dominant forms of Parkinson's Disease (PD). METHODS: Two families of Turkish origins with PD were studied; the SNCA coding region was analyzed by Sanger sequencing, and by whole exome sequencing (WES) in the index patient of the first and the second family, respectively. Co-segregation studies and haplotype analysis across the SNCA locus were carried out. Functional studies included in vitro thioflavin-T aggregation assay and in silico structural modelling of the alpha-synuclein (α-syn) protein. RESULTS: We identified a novel heterozygous SNCA variant, c.215C > T (p.Thr72Met), segregating with PD in a total of four members in the two families. A shared haplotype across the SNCA locus was found among variant carriers, suggestive of a common ancestor. We next showed that the Thr72Met α-syn displays enhanced aggregation in-vitro, compared to the wild-type species. In silico analysis of a tetrameric α-syn structural model revealed that Threonine 72 lies in the tetrameric interface, and substitution with the much larger methionine residue could potentially destabilize the tetramer. CONCLUSION: We present clinical, genetic, and functional data supporting a causative role of the SNCA c.215C > T (p.Thr72Met) variant in familial PD. Testing for this variant in patients with PD, especially of Turkish origin, might detect additional carriers. Further functional analyses might offer new insights into the shared biochemical properties of the PD-causing SNCA missense variants, and how they lead to neurodegeneration.

MCF7 Spheroid Development: New Insight about Spatio/Temporal Arrangements of TNTs, Amyloid Fibrils, Cell Connections, and Cellular Bridges.

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme.