Culprit Genes Research Articles

Minning of Immuno-mitotic and GABAergic genes as potential biomarkers of glioblastoma: An integrated transcriptomic analysis

Glioblastoma (GBM) is a highly lethal Central Nervous System (CNS) tumor prevalent in both adults and children, exhibiting elevated rates of mortality and morbidity. Due to the heterogenous nature of GBM, coupled with its nonspecific symptoms underscore the imperative for innovative biomarkers to enhance prognosis and the development of efficacious therapeutic interventions. This bioinformatics study seeks to elucidate the culprit genes, both up-regulated and down-regulated, within the context of their functional relevance, through a comparative analysis of gene expression profiles in GBM and normal brain tissues. Deregulated genes were identified from two Gene Expression Omnibus (GEO) datasets, employing the GEO2R tool to analyze expression data from normal and GBM tissues. Subsequently, differences in expression of genes (DEGs) through functional enrichment analysis were conducted by DAVID to discern their functional implications. Further, Protein-Protein Interaction (PPI) networks were constructed to identify hub genes among the selected up-regulated and down-regulated genes, employing various bioinformatics tools. The impact of the selected hub genes on patient overall survival was investigated using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Notably, the up-regulated hub genes KIF2C and TTK exhibited significant correlations with overall survival, implicating their potential as immuno-mitotic biomarkers. Conversely, GAD2, the sole down-regulated hub gene, emerged as a promising molecular target for GBM, given its association with GABAergic signaling and amino acid metabolism. Consequently, these findings suggest that KIF2C and TTK may serve as immune-mitotic biomarkers, while GAD2 could be explored as a molecular target for GBM therapy. Nevertheless, additional research is essential to unravel the precise mechanistic underpinnings of GBM.

Advances in Multi-Modality Imaging in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by abnormal growth of the myocardium with myofilament disarray and myocardial hyper-contractility, leading to left ventricular hypertrophy and fibrosis. Where culprit genes are identified, they typically relate to cardiomyocyte sarcomere structure and function. Multi-modality imaging plays a crucial role in the diagnosis, monitoring, and risk stratification of HCM, as well as in screening those at risk. Following the recent publication of the first European Society of Cardiology (ESC) cardiomyopathy guidelines, we build on previous reviews and explore the roles of electrocardiography, echocardiography, cardiac magnetic resonance (CMR), cardiac computed tomography (CT), and nuclear imaging. We examine each modality’s strengths along with their limitations in turn, and discuss how they can be used in isolation, or in combination, to facilitate a personalized approach to patient care, as well as providing key information and robust safety and efficacy evidence within new areas of research.

Human Genetics of Tricuspid Atresia and Univentricular Heart.

Tricuspid atresia (TA) is a rare congenital heart condition that presents with a complete absence of the right atrioventricular valve. Because of the rarity of familial and/or isolated cases of TA, little is known about the potential genetic abnormalities contributing to this condition. Potential responsible chromosomal abnormalities were identified in exploratory studies and include deletions in 22q11, 4q31, 8p23, and 3p as well as trisomies 13 and 18. In parallel, potential culprit genes include the ZFPM2, HEY2, NFATC1, NKX2-5, MYH6, and KLF13 genes. The aim of this chapter is to expose the genetic components that are potentially involved in the pathogenesis of TA in humans. The large variability in phenotypes and genotypes among cases of TA suggests a genetic network that involves many components yet to be unraveled.

Lessons of Nature in Trisomy-8 MDS and AML: Determinants of Clonal Drive

The clinical consequences of trisomy 8 (+8) are reflected by its categorization as an intermediate risk cytogenetic abnormality in AML while in MDS it has a more favorable impact. Gain of chromosome (chr.) 8 is a frequent event in other hematologic disorders and its prognostic role seems to be dependent on disease context. Some molecular lesions ( RUNX1, ASXL1) are distinctive of +8 AML and important in delineating its clinical phenotype and prognosis. However, the intrinsic molecular drivers contributing to clonal pathogenesis of myeloid neoplasia (MN) as repercussion of gain of chr.8 remain unclear. We stipulated that the study of clinical, genomic, and transcriptomic features of +8 MN will provide important clues to the pathogenesis of +8 and help refine its clinical implications in a specific clinical context. NGS and clinical data from a cohort of 3971 MDS and 3973 AML patients were reviewed, including normal karyotype MDS/AML (NK MDS or AML), isolated +8 (iso +8 MDS/AML), and non-isolated +8 (non-iso +8 MDS/AML). In addition, RNASeq data were available in a subcohort of patients (82: +8; 617 NK MDS). Expression of chr.8 genes was studied to unmask culprit genes in +8 MN. Compared to NK-AML, iso +8 AML patients were older (76% aged >60 years, P= .0005) and had more dysplastic than hyperproliferative phenotype as they presented with lower WBC (median: 9.5 vs 15.2x10 9/L, P= .008) and platelet counts (median: 49 vs 64 x10 9/L, P= .0002). Iso +8 AML had higher percentages of secondary AML (sAML, 17 vs 11%, P= .001) arising from antecedent MDS (14 vs 9%, P= .002) vs NK AML. As expected, survival analysis showed iso +8-AML to have worse median overall survival (mOS) vs NK AML (20 vs 32 months, P= .0001). Yet, when sub-classifying AML based on disease ontogeny (primary vs secondary), a significant mOS difference was only seen in primary AML (21.5 vs 34.5 months, P= .0002; in sAML, 14.7 vs 24.3 months, P= .3). As for molecular associations, a variety of mutations were more frequent in iso +8 AML vs NK AML, including: lineage transcriptional factors ( RUNX1, 33 vs 17%, P< .0001), chromatin modifiers ( ASXL1 37 vs 13%, P< .0001; EZH2 10 vs 3.4%, P< .0001), splicing factors ( SRSF2 30 vs 13%, P< .0001; U2AF1, 12 vs 3%, P< .0001) and cohesin genes ( STAG2, 13 vs 7%, P= .02). In contrast, mutations in FLT3 (33 vs 42%, = .01), DNMT3A (24 vs 35%, P=.002), NPM1 (14 vs 44%, P<.0001) and CEBPA (7 vs 12%, P= .02) were more frequent in NK AML. We then focused on the iso +8 MDS vs NK MDS groups, and interestingly, found that ASXL1 (52 vs 19%, P< .0001), EZH2 (25 vs 4, P< .0001), RUNX1 (29 vs 12%, P= .0009), and STAG2 (27 vs 6%, P< .0001) were more frequently found in the +8 MDS compared to NK-MDS (similar patten to AML), whereas SRSF2 (19 vs 18%, P= .9) and U2AF1 (9 vs 6%, P=.7) were similar in both. The comparison of AML and MDS then showed that iso +8 AML was more commonly associated with mutations in FLT3 (33 vs 3%, P< .0001), IDH1 (17 vs 5%, P= .03), and IDH2 (25 vs 3%, P< .0001), while iso +8-MDS had higher odds of SF3B1 (19 vs 4%, P= .001) ( Fig1A). We then investigated whether specific transcriptomic features may play a role in +8 MN pathogenesis by targeting the transcriptome of 412 chr.8 genes ( Fig1B). After adjusting for zygosity, we identified 186 genes correlating the change in expression levels with clonality. We further interrogated genes possibly eliciting oncogenic potential with log2 fold change (log2FC) ratio of at least 1.25 to reflect an extra chr.8 copy. We found higher mRNA expression levels of 13 putative culprit genes of known oncogenic potential in iso +8 MDS vs NK MDS including the anti-apoptotic protein BAG4 (LogFC 7.1 vs 5.4, P< .0001), possibly conferring growth advantage, toll-like receptor LY96 (LogFC 3.3 vs 2.0, P< .0001),the tyrosine protein kinase LYN (LogFC 8.4 vs 6.7, P< .0001), human oncogene WHSC1L1/NSD3 (LogFC 8.1 vs 6.5, P< .0001)and long non-coding RNA, PVT1 derived from an 8q24 intergenic region (LogFC 4.0 vs 2.2, P< .0001). In summary, iso +8 MN represents a distinct entity defined by older age and dysplastic phenotype, likely due to genotypic enrichment in RUNX1 mutations, while inversely associated with proliferation genes ( e.g., FLT3 and NPM1 in NK). The key culprit oncogenes implicated in +8 MN may be located on the extra chr.8 itself. Indeed, we found transcriptomic changes pertinent to +8 MDS whose role in driving leukemogenesis and interaction with specific myeloid drivers needs to be confirmed by future experimentations on cellular and murine models.

Identification of Culprit Genes for Different Diseases by Analyzing Microarray Data.

The identification of disease-causing genes is the first and most important step toward understanding the biological mechanisms underlying a disease. Microarray analysis is one such powerful method that is widely used to identify genes that are expressed differently in two or more conditions (disease vs. normal). Because of its large library of statistical R packages and user-friendly interface, the R programming language provides a platform for microarray analysis. In this chapter, we will go over how to identify disease-causing culprit genes from the raw microarray data, using various packages of R programming. The pipeline overviews the steps in microarray analysis, such as data pre-processing, normalization, and statistical analysis using visualization techniques such as heatmaps, box plots, and so on. To better understand the function of the altered genes, gene ontology and pathway analysis are performed.

Characterization of infectious and non-infectious gastrointestinal disease in common variable immunodeficiency: analysis of 114 patient cohort.

Common Variable Immunodeficiency (CVID), a complex primary immunodeficiency syndrome defined by defective B cell responses to infection and vaccination, has heterogeneous clinical manifestations. Gastrointestinal (GI) complications in CVID, both infectious and non-infectious, can cause significant impairment leading to malabsorption and frank malnutrition. In order to better characterize the spectrum of GI disease associated with CVID, we describe 114 patients with GI disease (15.6%) from our 728 patient single center CVID cohort. Norovirus, Giardia and Cytomegalovirus were the most frequently isolated infectious pathogens. CVID enteropathy was the most encountered GI diagnosis based on endoscopy, with only a minority of patients having Crohn's disease (6.1%) or ulcerative colitis/proctitis (4.5%). Concurrent autoimmunity (30.7%), lung disease (18.4%) and malignancy (8.7%) were also present in significant proportion of subjects. Lastly, 16 of 47 (34%) who underwent whole exome sequencing demonstrated a culprit gene defect associated with CVID.

Establishment of Pancreatic Beta Cell-specific Gene Knockout System Based on CRISPR-Cas9 Technology with AAV8-mediated gRNA Delivery.

The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout, including pancreatic beta cells associated with the pathogenesis of diabetes mellitus. However, it is expensive and time-consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre-expression mice. We establish a βCas9 system with mice expressing Cas9 in pancreatic beta cells and adeno-associated virus 8 (AAV8) -mediated gRNA delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-LoxP-Stop-LoxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucosetolerant βCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in beta cells. We also show significant beta cell-specific gene knockout efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administer AAV8 to βCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4 (MODY4). As reported previously, we demonstrate that those mice show glucose intolerance with trans-differentiation of Pdx1 knockout beta cells into glucagonexpressing cells. We successfully generate a convenient beta cell-specific gene knockout system with βCas9 mice and AAV8-mediated gRNA delivery.

A harmful MYH11 variant detected in a family with thoracic aortic dissection and patent ductus arteriosus.

Thoracic aortic dissection (TAD) is an important cause of sudden cardiac death and is characterized by high morbidity, mortality, and a poor prognosis. Patent ductus arteriosus (PDA) is a common congenital heart disease. The pathogenesis of both TAD and PDA has been reported to be related to genetic factors. The MYH11 gene, which encodes myosin heavy chain 11, has been reported in individuals with both TAD and PDA. Herein, we first detected a harmful MYH11 missense variant (c. T3728C, p. L1243P) in a TAD and PDA family. This missense variant co-segregated with the TAD/PDA phenotype in this family of four individuals, providing evidence of its harmfulness. Histopathological examinations revealed the presence of fragmented, broken, and lessened elastic fibers and the deposition of proteoglycans in the median of aortic dissection. Moreover, the immunofluorescence results showed that the labeled MYH11 protein in the tissue of the aortic dissection was weaker than that in the normal aorta. We present this familial case to stress the necessity of postmortem genetic testing in such cases among forensic practices. Identifying those culprit gene variants can direct effective genetic counseling and personalized health management in family members (especially first-degree relatives) with high-risk genotypes.

New molecular targets in canine hemangiosarcoma-Comparative review and future of the precision medicine.

Human angiosarcoma and canine hemangiosarcoma reveal similarities not only in their aggressive clinical behaviour, but especially in molecular landscape and genetic alterations involved in tumorigenesis and metastasis formation. Currently, no satisfying treatment that allows for achieving long overall survival or even prolonged time to progression does not exist. Due to the progress that has been made in targeted therapies and precision medicine the basis for a new treatment design is to uncover mutations and their functions as possible targets to provide tailored drugs for individual cases. Whole exome or genome sequencing studies and immunohistochemistry brought in the last few years important discoveries and identified the most common mutations with probably crucial role in this tumour development. Also, despite a lack of mutation in some of the culprit genes, the cancerogenesis cause may be buried in main cellular pathways connected with proteins encoded by those genes and involving, for example, pathological angiogenesis. The aim of this review is to highlight the most promising molecular targets for precision oncology treatment from the veterinary perspective aided by the principles of comparative science. Some of the drugs are only undergoing laboratory in vitro studies and others entered the clinic in the management of other cancer types in humans, but those used in dogs with promising responses have been mentioned as priorities.

Genetic cold cases: lessons from solving complex congenital limb disorders.

Congenital genetic disorders affecting limb morphology in humans and other mammals are particularly well described, due to both their rather high frequencies of occurrence and the ease of their detection when expressed as severe forms. In most cases, their molecular and cellular etiology remained unknown long after their initial description, often for several decades, and sometimes close to a century. Over the past 20 yr, however, experimental and conceptual advances in our understanding of gene regulation, in particular over large genomic distances, have allowed these cold cases to be reopened and, eventually, for some of them to be solved. These investigations led not only to the isolation of the culprit genes and mechanisms, but also to the understanding of the often complex regulatory processes that are disturbed in such mutant genetic configurations. Here, we present several cases in which dormant regulatory mutations have been retrieved from the archives, starting from a historical perspective up to their molecular explanations. While some cases remain open, waiting for new tools and/or concepts to bring their investigations to an end, the solutions to others have contributed to our understanding of particular features often found in the regulation of developmental genes and hence can be used as benchmarks to address the impact of noncoding variants in the future.

Pharmacologic Management of Monogenic and Very Early Onset Inflammatory Bowel Diseases.

Inflammatory bowel disease (IBD) is treated with a variety of immunomodulating and immunosuppressive therapies; however, for the majority of cases, these therapies are not targeted for specific disease phenotypes. Monogenic IBD with causative genetic defect is the exception and represents a disease cohort where precision therapeutics can be applied. With the advent of rapid genetic sequencing platforms, these monogenic immunodeficiencies that cause inflammatory bowel disease are increasingly being identified. This subpopulation of IBD called very early onset inflammatory bowel disease (VEO-IBD) is defined by an age of onset of less than six years of age. Twenty percent of VEO-IBDs have an identifiable monogenic defect. The culprit genes are often involved in pro-inflammatory immune pathways, which represent potential avenues for targeted pharmacologic treatments. This review will provide an overview of the current state of disease-specific targeted therapies, as well as empiric treatment for undifferentiated causes of VEO-IBD.

What Kept Back on the Mirror of COVID-19-Related Acute Transverse Myelitis? A Genetic Background!

COVID-19-associated acute transverse myelitis (ATM) cases have been reported worldwide. Nevertheless, Iran, Italy, and the USA are the most affected countries, witnessing the possibility that genetic factors might be associated with this susceptibility. The genetic variants of the coronavirus-2 entry mechanisms and host innate immune response-related genes like interferons, interleukins, Toll-like receptors, human leukocyte antigens, blood groups, and some risk loci may be accountable. This study describes the compatibility of the geographical distribution between ATM and the Neanderthal core haplotype that confers risk for severe COVID-19 and some possible culprit genes.

Trisomy-6: The Ying and Yang of Chromosome 6 Aberrations

Karyotypic aberrations of chromosome 6 (chr.6), include duplications (trisomy 6; +6), deletion of its short arm (del6p), uniparental disomy 6 (UPD6p), as well as various microduplications and microdeletions. Among many important genes, the HLA locus is located on 6p, and loss of heterozygosity and allelic loss have been described as means of immune escape in immune-mediated aplastic anemia (AA) and in relapse of myeloid neoplasia following allogeneic bone marrow transplant. The pathogenesis of duplication of chr.6 appears more complex and might involve deletion of a putative tumor suppressor gene (TSG) causing duplication of the HLA locus with increased HLA expression. We have encountered several patients with +6 who inspired us to investigate the clinical and molecular phenotypes of +6. A total of 29 +6 MDS patients were identified, of whom 16 had isolated +6 (Iso +6). When compared to normal karyotype (NK) MDS (n=420), median age was younger in Iso +6 (70 vs 42 years, P<.0001). While no differences were noted for white blood counts, Iso +6 patients had lower platelets (median 27 vs. 112 x 103/uL, P=.19), were more frequently anemic (median hemoglobin 8.4 vs. 9.5 g/dL, P=.04) and showed higher frequency of hypocellularity at marrow evaluation (62.5% vs. 9.5%, P=0.0016) as opposed to NK MDS. Indeed, 67% of Iso +6 patients had a clinical picture reminiscent of AA or MDS phenotype which might have evolved from an antecedent AA phase. However, 67% of Iso +6 MDS had high risk MDS (IPSS-R ≥3.5) and 37% transformed to AML. Thus, our cohort indicated a phenotypic dichotomy which include hypocellular MDS/AA and high-risk disease with a poor prognosis. Of all +6 MDS, mutations most commonly affected TP53 (69%) followed by EZH2 (19%). Recurrent copy number variations (CNV) such as micro-duplications and microdeletions may be used to identify culprit genes. Indeed, microdeletions involving HLA have previously hinted towards the pathogenic role of HLA deletions. We obtained CNV data and compared them to conventional germline chromosome 6 CNV gains publicly available in the Database of Genomic Variants (DGV). Genes pertaining to significantly duplicated segments were then identified using the UCSC Genome Browser (https://genome.ucsc.edu/cgi-bin/hgGateway). In our cohort, in addition to 833 cases with del6p, we have identified microduplications involving 6p locus in 115 (11.5%) NK MDS cases. Overall, a total of 1198 6p copy gains were found in these patients, ranging in size 0.09-7.8Mb and with a median of 0.12Mb. The genomic architecture of each segment was investigated using the UCSC Genome Browser, from which only complete gene duplications that mirror an extra chromosomal copy were considered. Complete duplications were most commonly seen in loci containing genes of DUSP22 (12.2%) and most importantly HLA-DRB1 (10.4%), HLA-DRB5 (10.4%), HLA-DQA1 (9.6%) and MICA (9.6%). All of these five genes were significantly enriched in NK MDS as compared to healthy controls (DUSP22 3% P<0.0001, HLA-DRB1 0.4% P<0.0001, HLA-DRB5 0.5% P<0.0001, HLA-DQA1 0.5% P<0.0001 and MICA 0.2% P<0.0001) (Fig 1). Interestingly, while checking the Cosmic Cancer Browser database (https://cancer.sanger.ac.uk), we noticed that copy gains involving HLA-DRB1 and HLA-DRB5 were also more common in primary AML than in healthy controls from DGV (3.6 vs 0.4%, P<0.0001 and 3.6 vs 0.5%, P<0.0001, respectively). While the involvement of the HLA genes hints towards an immune-mediated mechanism underlying the pathogenesis of +6 MDS, further involvement in primary AML may suggest a common mechanism through which CN gains of the HLA locus promote myelo-neoplasia. We stipulate that while clonal deletion or UPD of HLA locus can be a result of immune pressure as seen in AA and during GvL, clonal duplication may lead to increased autoantigens presentation beyond the TCR activation threshold, triggering immune reactions ultimately resulting in hypocellular marrow and AA-like presentation. Alternatively, +6 and related lesions may lead to risk allele duplication and consequential more efficient presentation of a specific antigenic peptide. In more advanced diseases, deletion of TSG may also play a role, with progression reflecting a somatic pressure to overcome immune inhibition. HLA constellation and mutational studies will be presented in the context of the findings of 6p duplications and microdeletion in MDS and AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Pathogenic CDH3 Variant underlying Heredity Hypotrichosis Simplex detected by Whole-Exome Sequencing (WES)-A Case Report.

Heredity hypotrichosis simplex (HHS) is a rare nonsyndromic disease form of hypotrichosis simplex (HS) characterized by progressive hair follicle (HF) miniaturization. It is usually inherited in an autosomal dominant manner. The differential diagnosis of HHS and the treatments remain challenging despite recent advancement. In this report, we describe a 19-yr-old female affected with HHS alongside most of her family members. Whole-exome sequencing (WES) was performed for some of the family members to unravel the culprit gene involved in HHS phenotype and ascertain the dermatological examination that was done to classify the phenotypes of the disease. A novel pathogenic variant in the CDH3 gene (p.Ser223GlyfsTer4) was identified as a plausible disease-causing variant for HHS. This is the first report to associate CDH3 variants with a HHS phenotype without macular degeneration using WES. WES is an important tool for genotype–phenotype correlation, precision in diagnosis, and in-depth understanding of the disease mechanisms, leading to possible novel therapeutic targets treatment and better patient outcomes.

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling

BackgroundRare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. ObjectivesTo characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. Patients/MethodsIndex case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. ResultsFive family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels. ConclusionsThis study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia.

Cerebellum Dysfunction in Patients With PRRT2-Related Paroxysmal Dyskinesia.

The main culprit gene for paroxysmal kinesigenic dyskinesia, characterized by brief and recurrent attacks of involuntary movements, is PRRT2. The location of the primary dysfunction associated with paroxysmal dyskinesia remains a matter of debate and may vary depending on the etiology. While striatal dysfunction has often been implicated in these patients, evidence from preclinical models indicates that the cerebellum could also play a role. We aimed to investigate the role of the cerebellum in the pathogenesis of PRRT2-related dyskinesia in humans. We enrolled 22 consecutive right-handed patients with paroxysmal kinesigenic dyskinesia with a pathogenic variant of PRRT2 and their matched controls. Participants underwent a multimodal neuroimaging protocol. We recorded anatomic and diffusion-weighted MRI, as well as resting-state fMRI, during which we tested the aftereffects of sham and repetitive transcranial magnetic stimulation applied to the cerebellum on endogenous brain activity. We quantified the structural integrity of gray matter using voxel-based morphometry, the structural integrity of white matter using fixel-based analysis, and the strength and direction of functional cerebellar connections using spectral dynamic causal modeling. Patients with PRRT2 had decreased gray matter volume in the cerebellar lobule VI and in the medial prefrontal cortex, microstructural alterations of white matter in the cerebellum and along the tracts connecting the cerebellum to the striatum and the cortical motor areas, and dysfunction of cerebellar motor pathways to the striatum and the cortical motor areas, as well as abnormal communication between the associative cerebellum (Crus I) and the medial prefrontal cortex. Cerebellar stimulation modulated communication within the motor and associative cerebellar networks and tended to restore this communication to the level observed in healthy controls. Patients with PRRT2-related dyskinesia have converging structural alterations of the motor cerebellum and related pathways with a dysfunction of cerebellar output toward the cerebello-thalamo-striato-cortical network. We hypothesize that abnormal cerebellar output is the primary dysfunction in patients with a PRRT2 pathogenic variant, resulting in striatal dysregulation and paroxysmal dyskinesia. More broadly, striatal dysfunction in paroxysmal dyskinesia might be secondary to aberrant cerebellar output transmitted by thalamic relays in certain disorders. ClinicalTrials.gov identifier: NCT03481491.

Characterization of regulatory roles of genetic signals curated from more than 200 GWA studies in the Alzheimer's Disease Variant Portal (ADVP).

The Alzheimer's Disease Variant Portal (ADVP, https://advp.niagads.org) is a curated and harmonized interactive platform providing the research community with a unified way to access and visualize the largest up-to-date collection of Alzheimer's disease (AD) genetic findings from >200 genome-wide association studies (GWASs). In this study, we aim to characterize the regulatory roles and mechanisms underlying these genetic signals in AD. We analyzed all Caucasian genome-wide significant signals in ADVP (348 genetic variants and 186 loci) using FILER (Kuksa et al., 2021) and SparkINFERNO (Kuksa et al., 2020). The SparkINFERNO high throughput system analyses framework is coupled with the FILER (https://lisanwanglab.org/FILER), a rich resource of >50,000 harmonized functional genomic datasets across many different sources including ENCODE, FANTOM5, GTEx, Roadmap, DASHR2 and others. With this systematic, hypothesis-free analysis approach, we analyzed and ranked relevant tissue categories by quantifying the significance and enrichment of tissue-specific enhancer overlaps with ADVP variants compared to the empirical overlap distribution for matching background (non-AD) variants. Further integration with the AMP-AD expression quantitative trait loci (eQTL) datasets from three brain regions (dorsolateral prefrontal cortex, cerebellum and temporal cortex), enable us to discover the potentially causal genes affected by these variants. The ADVP variants were enriched in regulatory elements profiled by FANTOM5 and Roadmap Epigenomics (ChromHMM). Top tissues enriched in Roadmap enhancers include blood (OR=3.7), digestive (OR=3.5), brain (OR=2.8), and skeletal muscles (OR=2.6). 31% of the analyzed ADVP variants were identified as significant eQTLs in at least one of the three AMP-AD eQTL datasets (FDR<0.01), and 32% among these were eQTLs in all three brain regions. Altogether, these variants targeted 130 genes (including HLA regions), of which 31 were also the nearest genes reported in ADVP. These target genes (e.g. ACE, PVR, KANSL1, NARS2) were enriched in cell junction organization, DNA repair (nucleotide-excision repair) and acetyltransferase activity (cholinergic system), which recapitulated some of the molecular and cellular mechanisms underlying the pathogenesis of AD. Conducted analysis of genetic associations across >200 GWASs in ADVP and identified known AD culprit genes, relevant tissue/cell type categories, and regulatory mechanisms.

Whole-Transcriptome Analysis by RNA Sequencing for Genetic Diagnosis of Mendelian Skin Disorders in the Context of Consanguinity

Among the approximately 8000 Mendelian disorders, >1000 have cutaneous manifestations. In many of these conditions, the underlying mutated genes have been identified by DNA-based techniques which, however, can overlook certain types of mutations, such as exonic-synonymous and deep-intronic sequence variants. Whole-transcriptome sequencing by RNA sequencing (RNA-seq) can identify such mutations and provide information about their consequences. We analyzed the whole transcriptome of 40 families with different types of Mendelian skin disorders with extensive genetic heterogeneity. The RNA-seq data were examined for variant detection and prioritization, pathogenicity confirmation, RNA expression profiling, and genome-wide homozygosity mapping in the case of consanguineous families. Among the families examined, RNA-seq was able to provide information complementary to DNA-based analyses for exonic and intronic sequence variants with aberrant splicing. In addition, we tested the possibility of using RNA-seq as the first-tier strategy for unbiased genome-wide mutation screening without information from DNA analysis. We found pathogenic mutations in 35 families (88%) with RNA-seq in combination with other next-generation sequencing methods, and we successfully prioritized variants and found the culprit genes. In addition, as a novel concept, we propose a pipeline that increases the yield of variant calling from RNA-seq by concurrent use of genome and transcriptome references in parallel. Our results suggest that "clinical RNA-seq" could serve as a primary approach for mutation detection in inherited diseases, particularly in consanguineous families, provided that tissues and cells expressing the relevant genes are available for analysis.

Genome wide association analysis in dilated cardiomyopathy reveals two new key players in systolic heart failure on chromosomes 3p25.1 and 22q11.23

Dilated cardiomyopathy (DCM) is a major cause of systolic heart failure and therefore a major public health issue. Our objective was to better understand the genetic bases of dilated cardiomyopathy. We conducted a 1000G based genome-wide association study for 9,152,885 SNPs on 2719 sporadic DCM cases and 4440 controls of European origin followed by a replication step. We then sought for the most likely culprit genes at the new replicated loci through a dedicated strategy including in silico data mining (including tissue specific gene expressions, expression and methylation quantitative trait loci) as well as functional 4C-sequencing analysis on iPSC-derived cardiomyocytes ( Fig. 1 ). We identified two new DCM loci, on chromosome 3p25.1 (lead SNP rs62232870, P = 8.7 × 10 −11 and 7.7 × 10 −4 in the discovery and replication steps, respectively) and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10 −8 and 1.4 × 10 −3 , respectively), while confirming two previously identified ones, BAG3 and HSPB7. A Genetic Risk Score was built from the number of risk allele at these four loci and revealed an increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). At chr3p25, our selection strategy pinpointed SLC6A6 as the most likely culprit gene. SLC6A6 encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, the same strategy strongly suggested SMARCB1 as the best candidate gene. This study provides new insights in the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure, with the potential for a therapeutic perspective especially through taurine modulation.

Inherited Platelet Disorders: An Updated Overview.

Platelets play a major role in hemostasis as ppwell as in many other physiological and pathological processes. Accordingly, production of about 1011 platelet per day as well as appropriate survival and functions are life essential events. Inherited platelet disorders (IPDs), affecting either platelet count or platelet functions, comprise a heterogenous group of about sixty rare diseases caused by molecular anomalies in many culprit genes. Their clinical relevance is highly variable according to the specific disease and even within the same type, ranging from almost negligible to life-threatening. Mucocutaneous bleeding diathesis (epistaxis, gum bleeding, purpura, menorrhagia), but also multisystemic disorders and/or malignancy comprise the clinical spectrum of IPDs. The early and accurate diagnosis of IPDs and a close patient medical follow-up is of great importance. A genotype–phenotype relationship in many IPDs makes a molecular diagnosis especially relevant to proper clinical management. Genetic diagnosis of IPDs has been greatly facilitated by the introduction of high throughput sequencing (HTS) techniques into mainstream investigation practice in these diseases. However, there are still unsolved ethical concerns on general genetic investigations. Patients should be informed and comprehend the potential implications of their genetic analysis. Unlike the progress in diagnosis, there have been no major advances in the clinical management of IPDs. Educational and preventive measures, few hemostatic drugs, platelet transfusions, thrombopoietin receptor agonists, and in life-threatening IPDs, allogeneic hematopoietic stem cell transplantation are therapeutic possibilities. Gene therapy may be a future option. Regular follow-up by a specialized hematology service with multidisciplinary support especially for syndromic IPDs is mandatory.