Phenotypic analysis of ataxia in spinocerebellar ataxia type 6 mice using DeepLabCut.

This study emphasizes the benefits of open-source software such as DeepLabCut (DLC) and R to automate, customize and enhance data analysis of motor behavior. We recorded 2 different spinocerebellar ataxia type 6 mouse models while performing the classic beamwalk test, tracked multiple body parts using the markerless pose-estimation software DLC and analyzed the tracked data using self-written scripts in the programming language R. The beamwalk analysis script (BAS) counts and classifies minor and major hindpaw slips with an 83% accuracy compared to manual scoring. Nose, belly and tail positions relative to the beam, as well as the angle at the tail base relative to the nose and tail tip were determined to characterize motor deficits in greater detail. Our results found distinct ataxic abnormalities such as an increase in major left hindpaw slips and a lower belly and tail position in both SCA6 ataxic mouse models compared to control mice at 18 months of age. Furthermore, a more detailed analysis of various body parts relative to the beam revealed an overall lower body position in the SCA684Q compared to the CT-longQ27PC mouse line at 18 months of age, indicating a more severe ataxic deficit in the SCA684Q group.

Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research.

Genome editing, notably CRISPR (cluster regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), has revolutionized genetic engineering allowing for precise targeted modifications. This technique's combination with human induced pluripotent stem cells (hiPSCs) is a particularly valuable tool in cerebral organoid (CO) research. In this study, CRISPR/Cas9-generated fluorescently labeled hiPSCs exhibited no significant morphological or growth rate differences compared with unedited controls. However, genomic aberrations during gene editing necessitate efficient genome integrity assessment methods. Optical genome mapping, a high-resolution genome-wide technique, revealed genomic alterations, including chromosomal copy number gain and losses affecting numerous genes. Despite these genomic alterations, hiPSCs retain their pluripotency and capacity to generate COs without major phenotypic changes but one edited cell line showed potential neuroectodermal differentiation impairment. Thus, this study highlights optical genome mapping in assessing genome integrity in CRISPR/Cas9-edited hiPSCs emphasizing the need for comprehensive integration of genomic and morphological analysis to ensure the robustness of hiPSC-based models in cerebral organoid research.

Attenuated huntingtin gene CAG nucleotide repeat size in individuals with Lynch syndrome.

DNA mismatch repair (MMR) is thought to contribute to the onset and progression of Huntington disease (HD) by promoting somatic expansion of the pathogenic CAG nucleotide repeat in the huntingtin gene (HTT). Here we have studied constitutional HTT CAG repeat size in two cohorts of individuals with Lynch syndrome (LS) carrying heterozygous loss-of-function variants in the MMR genes MLH1 (n = 12/60; Lund cohort/Bochum cohort, respectively), MSH2 (n = 15/88), MSH6 (n = 21/23), and controls (n = 19/559). The sum of CAG repeats for both HTT alleles in each individual was calculated due to unknown segregation with the LS allele. In the larger Bochum cohort, the sum of CAG repeats was lower in the MLH1 subgroup compared to controls (MLH1 35.40 CAG repeats ± 3.6 vs. controls 36.89 CAG repeats ± 4.5; p = 0.014). All LS genetic subgroups in the Bochum cohort displayed lower frequencies of unstable HTT intermediate alleles and lower HTT somatic CAG repeat expansion index values compared to controls. Collectively, our results indicate that MMR gene haploinsufficiency could have a restraining impact on constitutional HTT CAG repeat size and support the notion that the MMR pathway is a driver of nucleotide repeat expansion diseases.

H3 Acetylation-Induced Basal Progenitor Generation and Neocortex Expansion Depends on the Transcription Factor Pax6.

Enrichment of basal progenitors (BPs) in the developing neocortex is a central driver of cortical enlargement. The transcription factor Pax6 is known as an essential regulator in generation of BPs. H3 lysine 9 acetylation (H3K9ac) has emerged as a crucial epigenetic mechanism that activates the gene expression program required for BP pool amplification. In this current work, we applied immunohistochemistry, RNA sequencing, chromatin immunoprecipitation and sequencing, and the yeast two-hybrid assay to reveal that the BP-genic effect of H3 acetylation is dependent on Pax6 functionality in the developing mouse cortex. In the presence of Pax6, increased H3 acetylation caused BP pool expansion, leading to enhanced neurogenesis, which evoked expansion and quasi-convolution of the mouse neocortex. Interestingly, H3 acetylation activation exacerbates the BP depletion and corticogenesis reduction effect of Pax6 ablation in cortex-specific Pax6 mutants. Furthermore, we found that H3K9 acetyltransferase KAT2A/GCN5 interacts with Pax6 and potentiates Pax6-dependent transcriptional activity. This explains a genome-wide lack of H3K9ac, especially in the promoter regions of BP-genic genes, in the Pax6 mutant cortex. Together, these findings reveal a mechanistic coupling of H3 acetylation and Pax6 in orchestrating BP production and cortical expansion through the promotion of a BP gene expression program during cortical development.

Association of the neonatal Fc receptor promoter variable number of tandem repeat polymorphism with immunoglobulin response in patients with chronic inflammatory demyelinating polyneuropathy.

BACKGROUND AND PURPOSE: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune disease with humoral and cellular autoimmunity causing demyelination of peripheral nerves, commonly treated with intravenous immunoglobulins (IVIg). The neonatal Fc receptor (FcRn), encoded by the FCGRT gene, prevents the degradation of immunoglobulin G (IgG) by recycling circulating IgG. A variable number of tandem repeat (VNTR) polymorphism in the promoter region of the FCGRT gene is associated with different expression levels of mRNA and protein. Thus, patients with genotypes associated with relatively low FcRn expression may show a poorer treatment response to IVIg due to increased IVIg degradation. METHODS: VNTR genotypes were analyzed in 144 patients with CIDP. Patients' clinical data, including neurological scores and treatment data, were collected as part of the Immune-Mediated Neuropathies Biobank registry. RESULTS: Most patients (n = 124, 86%) were VNTR 3/3 homozygotes, and 20 patients (14%) were VNTR 2/3 heterozygotes. Both VNTR 3/3 and VNTR 2/3 genotype groups showed no difference in clinical disability and immunoglobulin dosage. However, patients with a VNTR 2 allele were more likely to receive subcutaneous immunoglobulins (SCIg) than patients homozygous for the VNTR 3 allele (25% vs. 9.7%, p = 0.02) and were more likely to receive second-line therapy (75% vs. 54%, p = 0.05). CONCLUSIONS: The VNTR 2/3 genotype is associated with the administration of SCIg, possibly reflecting a greater benefit from SCIg due to more constant immunoglobulin levels without lower IVIg levels between the treatment circles. Also, the greater need for second-line treatment in VNTR 2/3 patients could be an indirect sign of a lower response to immunoglobulins.

Symptomatic treatment options for Huntington's disease (guidelines of the German Neurological Society).

INTRODUCTION: Ameliorating symptoms and signs of Huntington's disease (HD) is essential to care but can be challenging and hard to achieve. The pharmacological treatment of motor signs (e.g. chorea) may favorably or unfavorably impact other facets of the disease phenotype (such as mood and cognition). Similarly, pharmacotherapy for behavioral issues may modify the motor phenotype. Sometimes synergistic effects can be achieved. In patients undergoing pragmatic polypharmacological therapy, emerging complaints may stem from the employed medications' side effects, a possibility that needs to be considered. It is recommended to clearly and precisely delineate the targeted signs and symptoms (e.g., chorea, myoclonus, bradykinesia, Parkinsonism, or dystonia). Evidence from randomized controlled trials (RCTs) is limited. Therefore, the guidelines prepared for the German Neurological Society (DGN) for German-speaking countries intentionally extend beyond evidence from RCTs and aim to synthesize evidence from RCTs and recommendations of experienced clinicians. RECOMMENDATIONS: First-line treatment for chorea is critically discussed, and a preference in prescription practice for using tiapride instead of tetrabenazine is noted. In severe chorea, combining two antidopaminergic drugs with a postsynaptic (e.g., tiapride) and presynaptic mode of action (e.g., tetrabenazine) is discussed as a potentially helpful strategy. Sedative side effects of both classes of compounds can be used to improve sleep if the highest dosage of the day is given at night. Risperidone, in some cases, may ameliorate irritability but also chorea and sleep disorders. Olanzapine can be helpful in the treatment of weight loss and chorea, and quetiapine as a mood stabilizer with an antidepressant effect. CONCLUSIONS: Since most HD patients simultaneously suffer from distinct motor signs and distinct psychiatric/behavioral symptoms, treatment should be individually adapted.

Differential diagnosis of chorea (guidelines of the German Neurological Society).

INTRODUCTION: Choreiform movement disorders are characterized by involuntary, rapid, irregular, and unpredictable movements of the limbs, face, neck, and trunk. These movements often initially go unnoticed by the affected individuals and may blend together with seemingly intended, random motions. Choreiform movements can occur both at rest and during voluntary movements. They typically increase in intensity with stress and physical activity and essentially cease during deep sleep stages. In particularly in advanced stages of Huntington disease (HD), choreiform hyperkinesia occurs alongside with dystonic postures of the limbs or trunk before they typically decrease in intensity. The differential diagnosis of HD can be complex. Here, the authors aim to provide guidance for the diagnostic process. This guidance was prepared for the German Neurological Society (DGN) for German-speaking countries. RECOMMENDATIONS: Hereditary (inherited) and non-hereditary (non-inherited) forms of chorea can be distinguished. Therefore, the family history is crucial. However, even in conditions with autosomal-dominant transmission such as HD, unremarkable family histories do not necessarily rule out a hereditary form (e.g., in cases of early deceased or unknown parents, uncertainties in familial relationships, as well as in offspring of parents with CAG repeats in the expandable range (27-35 CAG repeats) which may display expansions into the pathogenic range). CONCLUSIONS: The differential diagnosis of chorea can be challenging. This guidance prepared for the German Neurological Society (DGN) reflects the state of the art as of 2023.

Implications of specific lysine residues within ataxin-3 for the molecular pathogenesis of Machado-Joseph disease.

Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansion-dependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysine-free wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamine-expanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein.

Novel NUP98::ASH1L Gene Fusion in Acute Myeloid Leukemia Detected by Optical Genome Mapping.

Optical genome mapping (OGM) recently has demonstrated the potential to improve genetic diagnostics in acute myeloid leukemia (AML). In this study, OGM was utilized as a tool for the detection of genome-wide structural variants and disease monitoring. A previously unrecognized NUP98::ASH1L fusion was detected in an adult patient with secondary AML. OGM identified the fusion of NUP98 to Absent, Small, or Homeotic-Like Histone Lysine Methyltransferase (ASH1L) as result of a complex structural rearrangement between chromosomes 1 and 11. A pipeline for the measurement of rare structural variants (Rare Variant Pipeline, Bionano Genomics, San Diego, CA, USA) was used for detection. As NUP98 and other fusions are relevant for disease classification, this demonstrates the necessity for methods such as OGM for cytogenetic diagnostics in AML. Furthermore, other structural variants showed discordant variant allele frequencies at different time points over the course of the disease and treatment pressure, indicating clonal evolution. These results support OGM to be a valuable tool for primary diagnostics in AML as well as longitudinal testing for disease monitoring and deepening our understanding of genetically heterogenous diseases.

Structural architecture and brain network efficiency link polygenic scores to intelligence.

Intelligence is highly heritable. Genome-wide association studies (GWAS) have shown that thousands of alleles contribute to variation in intelligence with small effect sizes. Polygenic scores (PGS), which combine these effects into one genetic summary measure, are increasingly used to investigate polygenic effects in independent samples. Whereas PGS explain a considerable amount of variance in intelligence, it is largely unknown how brain structure and function mediate this relationship. Here, we show that individuals with higher PGS for educational attainment and intelligence had higher scores on cognitive tests, larger surface area, and more efficient fiber connectivity derived by graph theory. Fiber network efficiency as well as the surface of brain areas partly located in parieto-frontal regions were found to mediate the relationship between PGS and cognitive performance. These findings are a crucial step forward in decoding the neurogenetic underpinnings of intelligence, as they identify specific regional networks that link polygenic predisposition to intelligence.

Optical Genome Mapping for Cytogenetic Diagnostics in AML.

The classification and risk stratification of acute myeloid leukemia (AML) is based on reliable genetic diagnostics. A broad and expanding variety of relevant aberrations are structural variants beyond single-nucleotide variants. Optical Genome Mapping is an unbiased, genome-wide, amplification-free method for the detection of structural variants. In this review, the current knowledge of Optical Genome Mapping (OGM) with regard to diagnostics in hematological malignancies in general, and AML in specific, is summarized. Furthermore, this review focuses on the ability of OGM to expand the use of cytogenetic diagnostics in AML and perhaps even replace older techniques such as chromosomal-banding analysis, fluorescence in situ hybridization, or copy number variation microarrays. Finally, OGM is compared to amplification-based techniques and a brief outlook for future directions is given.

Optical Genome Mapping Reveals and Characterizes Recurrent Aberrations and New Fusion Genes in Adult ALL.

(1) Background: In acute lymphoblastic leukemia (ALL) the genetic characterization remains challenging. Due to the genetic heterogeneity of mutations in adult patients, only a small proportion of aberrations can be analyzed with standard routine diagnostics. Optical genome mapping (OGM) has recently opened up new possibilities for the characterization of structural variants on a genome-wide level, thus enabling simultaneous analysis for a broad spectrum of genetic aberrations. (2) Methods: 11 adult ALL patients were examined using OGM. (3) Results: Genetic results obtained by karyotyping and FISH were confirmed by OGM for all patients. Karyotype was redefined, and additional genetic information was obtained in 82% (9/11) of samples by OGM, previously not diagnosed by standard of care. Besides gross-structural chromosome rearrangements, e.g., ring chromosome 9 and putative isodicentric chromosome 8q, deletions in CDKN2A/2B were detected in 7/11 patients, defining an approx. 20 kb minimum region of overlap, including an alternative exon 1 of the CDKN2A gene. The results further confirm recurrent ALL aberrations (e.g., PAX5, ETV6, VPREB1, IKZF1). (4) Conclusions: Genome-wide OGM analysis enables a broad genetic characterization in adult ALL patients in one single workup compared to standard clinical testing, facilitating a detailed genetic diagnosis, risk-stratification, and target-directed treatment strategies.

Canavan's spongiform leukodystrophy (Aspartoacylase deficiency) with emphasis on sonographic features in infancy: description of a case report and review of the literature.

Canavan disease (CD; MIM 271,900) or spongy degeneration of the central nervous system (CNS) is a lethal, rare autosomal recessive leukodystrophy, first described in 1931 (Canavan in Arch Neurol Psychiatry 25: 299-308, 1931). The clinical presentation includes severe neurologic impairment and macrocephaly with onset of symptoms at the age of 3-5 months. Biochemical and genetic fundamentals of the disease are elucidated. Imaging diagnosis is principally based on MRI with important role of MR spectroscopy. We report the cerebral sonographic findings in a severely affected infant with CD: Diffuse hyperechogenicity and small multicystic changes of white matter as well as an inverted pattern of echogenicity between cortical gray and subcortical white matter. These findings are compared to to the few cases found in literature and to normal ultrasound examples. Finally, ultrasound and MRI imaging findings are correlated.

Evidences for Mutant Huntingtin Inducing Musculoskeletal and Brain Growth Impairments via Disturbing Testosterone Biosynthesis in Male Huntington Disease Animals.

Body weight (BW) loss and reduced body mass index (BMI) are the most common peripheral alterations in Huntington disease (HD) and have been found in HD mutation carriers and HD animal models before the manifestation of neurological symptoms. This suggests that, at least in the early disease stage, these changes could be due to abnormal tissue growth rather than tissue atrophy. Moreover, BW and BMI are reported to be more affected in males than females in HD animal models and patients. Here, we confirmed sex-dependent growth alterations in the BACHD rat model for HD and investigated the associated contributing factors. Our results showed growth abnormalities along with decreased plasma testosterone and insulin-like growth factor 1 (IGF-1) levels only in males. Moreover, we demonstrated correlations between growth parameters, IGF-1, and testosterone. Our analyses further revealed an aberrant transcription of testosterone biosynthesis-related genes in the testes of BACHD rats with undisturbed luteinizing hormone (LH)/cAMP/PKA signaling, which plays a key role in regulating the transcription process of some of these genes. In line with the findings in BACHD rats, analyses in the R6/2 mouse model of HD showed similar results. Our findings support the view that mutant huntingtin may induce abnormal growth in males via the dysregulation of gene transcription in the testis, which in turn can affect testosterone biosynthesis.

Calpain-mediated proteolysis as driver and modulator of polyglutamine toxicity.

Among posttranslational modifications, directed proteolytic processes have the strongest impact on protein integrity. They are executed by a variety of cellular machineries and lead to a wide range of molecular consequences. Compared to other forms of proteolytic enzymes, the class of calcium-activated calpains is considered as modulator proteases due to their limited proteolytic activity, which changes the structure and function of their target substrates. In the context of neurodegeneration and - in particular - polyglutamine disorders, proteolytic events have been linked to modulatory effects on the molecular pathogenesis by generating harmful breakdown products of disease proteins. These findings led to the formulation of the toxic fragment hypothesis, and calpains appeared to be one of the key players and auspicious therapeutic targets in Huntington disease and Machado Joseph disease. This review provides a current survey of the role of calpains in proteolytic processes found in polyglutamine disorders. Together with insights into general concepts behind toxic fragments and findings in polyglutamine disorders, this work aims to inspire researchers to broaden and deepen the knowledge in this field, which will help to evaluate calpain-mediated proteolysis as a unifying and therapeutically targetable posttranslational mechanism in neurodegeneration.