Christoph Preuss

Mutations in SGOL1 cause a novel cohesinopathy affecting heart and gut rhythm

The pacemaking activity of specialized tissues in the heart and gut results in lifelong rhythmic contractions. Here we describe a new syndrome characterized by Chronic Atrial and Intestinal Dysrhythmia, termed CAID syndrome, in 16 French Canadians and 1 Swede. We show that a single shared homozygous founder mutation in SGOL1, a component of the cohesin complex, causes CAID syndrome. Cultured dermal fibroblasts from affected individuals showed accelerated cell cycle progression, a higher rate of senescence and enhanced activation of TGF-β signaling. Karyotypes showed the typical railroad appearance of a centromeric cohesion defect. Tissues derived from affected individuals displayed pathological changes in both the enteric nervous system and smooth muscle. Morpholino-induced knockdown of sgol1 in zebrafish recapitulated the abnormalities seen in humans with CAID syndrome. Our findings identify CAID syndrome as a novel generalized dysrhythmia, suggesting a new role for SGOL1 and the cohesin complex in mediating the integrity of human cardiac and gut rhythm.

Exome sequencing identifies a novel variant in ACTC1 associated with familial atrial septal defect.

BACKGROUND The genetics of congenital heart disease (CHD) remain incompletely understood. Exome sequencing has been successfully used to identify disease-causing mutations in familial disorders in which candidate gene analyses and linkage mapping have failed. METHODS We studied a large family characterized by autosomal dominant isolated secundum atrial septal defect (ASD) (MIM No. 612794). Candidate gene resequencing and linkage analysis were uninformative. RESULTS Whole-exome sequencing of 2 affected family members identified 44 rare shared variants, including a nonsynonymous mutation (c.532A>T, p.M178L, NM_005159.4) in alpha-cardiac actin (ACTC1). This mutation was absent from 1834 internal controls as well as from the 1000 Genomes and the Exome Sequencing Project (ESP) databases, but predictions regarding its effect on protein function were divergent. However, p.M178L was the only rare mutation segregating with disease in our family. CONCLUSIONS Our results provide further evidence supporting a causative role for ACTC1 mutations in ASD. Massively parallel sequencing of the exome allows for the detection of novel rare variants causing CHD without the limitations of a candidate gene approach. When mutation prediction algorithms are not helpful, studies of familial disease can help distinguish rare pathologic mutations from benign variants. Consideration of the family history can lead to genetic insights into CHD.

Family Based Whole Exome Sequencing Reveals the Multifaceted Role of Notch Signaling in Congenital Heart Disease

Left-ventricular outflow tract obstructions (LVOTO) encompass a wide spectrum of phenotypically heterogeneous heart malformations which frequently cluster in families. We performed family based whole-exome and targeted re-sequencing on 182 individuals from 51 families with multiple affected members. Central to our approach is the family unit which serves as a reference to identify causal genotype-phenotype correlations. Screening a multitude of 10 overlapping phenotypes revealed disease associated and co-segregating variants in 12 families. These rare or novel protein altering mutations cluster predominantly in genes (NOTCH1, ARHGAP31, MAML1, SMARCA4, JARID2, JAG1) along the Notch signaling cascade. This is in line with a significant enrichment (Wilcoxon, p< 0.05) of variants with a higher pathogenicity in the Notch signaling pathway in patients compared to controls. The significant enrichment of novel protein truncating and missense mutations in NOTCH1 highlights the allelic and phenotypic heterogeneity in our pediatric cohort. We identified novel co-segregating pathogenic mutations in NOTCH1 associated with left and right-sided cardiac malformations in three independent families with a total of 15 affected individuals. In summary, our results suggest that a small but highly pathogenic fraction of family specific mutations along the Notch cascade are a common cause of LVOTO.

Genetics of Heart Failure in Congenital Heart Disease

Heart failure is a major problem in the patient with congenital heart disease. Normally interpreted as a sequela of surgical interventions or abnormal preoperative loading conditions, there is increasing evidence that congenital heart malformations and abnormal ventricular function can have the same underlying genetic cause. With the changing demographic characteristics and increasing complexity of care for patients with congenital heart disease, it can be anticipated that heart failure will be a rapidly growing concern in our field. In this article, we aim to give an overview of recent findings from mouse and human models that highlight shared pathways for the regulation of cardiac development and contractility, and their importance for medical care in the near future.

Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter ≥ 4.0 cm in adults, or a Z-score ≥ 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype.

Aortic Dilatation Associated With a De Novo Mutation in the SOX18 Gene: Expanding the Clinical Spectrum of Hypotrichosis-Lymphedema-Telangiectasia Syndrome

BACKGROUND We report a 13-year-old female patient followed since birth for multiple rare congenital defects, including hypotrichosis, telangiectasia, and severe dilatation of the ascending aorta. METHODS Comprehensive phenotype assessment throughout childhood included repeated echocardiographic measurements, evaluation of renal function, and immunohistochemical analysis of skin biopsy samples. Whole-exome sequencing was performed for the patient and both unaffected parents. RESULTS We identified a novel de novo mutation in the transcription factor SOX18 (c.481C>T:p.Gln161*) in the patient, which was absent in all unaffected family members. Echocardiography revealed early onset and progressive dilatation of the ascending aorta. Skin biopsy results confirmed the defects of the blood vasculature in the presence of intact lymphatic vessels. Assessment of renal function did not show any signs of renal problems or renal failure in the patient. CONCLUSIONS The genetic finding of a pathogenic SOX18 mutation enabled the diagnosis of the rare hypotrichosis-lymphedema-telangiectasia syndrome in our patient. The identification of a novel stop gain mutation in the SOX18 gene in association with dilatation of the aorta highlights the importance of this gene during the development of the circulatory system. Our study highlights the importance of whole-exome sequencing in the rapid identification of genes and gene mutations involved in rare conditions and thus expanding the knowledge and spectrum of clinical manifestations associated with them.

Epistatic interaction between the lipase-encoding genes Pnpla2 and Lipe causes liposarcoma in mice

Liposarcoma is an often fatal cancer of fat cells. Mechanisms of liposarcoma development are incompletely understood. The cleavage of fatty acids from acylglycerols (lipolysis) has been implicated in cancer. We generated mice with adipose tissue deficiency of two major enzymes of lipolysis, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), encoded respectively by Pnpla2 and Lipe. Adipocytes from double adipose knockout (DAKO) mice, deficient in both ATGL and HSL, showed near-complete deficiency of lipolysis. All DAKO mice developed liposarcoma between 11 and 14 months of age. No tumors occurred in single knockout or control mice. The transcriptome of DAKO adipose tissue showed marked differences from single knockout and normal controls as early as 3 months. Gpnmb and G0s2 were among the most highly dysregulated genes in premalignant and malignant DAKO adipose tissue, suggesting a potential utility as early markers of the disease. Similar changes of GPNMB and G0S2 expression were present in a human liposarcoma database. These results show that a previously-unknown, fully penetrant epistatic interaction between Pnpla2 and Lipe can cause liposarcoma in mice. DAKO mice provide a promising model for studying early premalignant changes that lead to late-onset malignant disease.

NOVEL MODELS OF LATE-ONSET ALZHEIMER’S DISEASE BASED ON GWAS

amyloid beta(Ab) and protecting nerve cells. Furthermore, 6MSITC has been reported to be absorbed a high rate in the body, which is considered to be about from 40 to 60%. Other isothiocyanate, as an analog of 6-MSITC, has been reported to permeate the brain blad barrier. Therefore, 6-MSITC can be considered as an effective compound for brain function. Methods: The doubleblinded clinical study was conducted using 6-MSITC (25 placebo, 25 subjects, 8 weeks ingestion), and the rat pheochromocytoma cell line, PC12, were used to study the synergistic effects for the neurite outgrowth, dopamine secretion and Ab toxicity by 6-MSITC, DHA, curcumin, isorhamnetin. Results:Significant results were obtained by the clinical test. Furthermore, we found that 6-MSITC reduced the toxicity from Ab, increased secretion of dopamine on nerve cells. 6-MSITC also has synergistic effects of DHA, curcumin, and ginkgo biloba compound, which are considered to be good compounds for improvement of brain function. Conclusions: Thus, 6-MSITC may be used a compound to prevent Alzheimer’s disease.

SYSTEMS BIOLOGY RANKING OF CANDIDATE ALZHEIMER’S DRIVER GENES IDENTIFIES NEW GENETIC DRIVERS OF ALZHEIMER’S DISEASE ETIOLOGY

Background: The standard approaches to understand gene expression and regulation in the brain include identification of differentially and co-expressed genes. To this end, the Accelerating Medicines Partnership Alzheimer’s Disease (AMP-AD) consortium has produced multiple, large RNA-seq datasets from several postmortem brain regions. Separately, the ENCODE project has produced DNAse Hypersensitivity (DHS) samples for various brain regions. We have integrated these large datasets into transcriptional regulatory networks (TRN), providing a directional and mechanistic list of putative transcription factors for nearly all expressed genes in the brain. Methods: We reprocessed all ENCODE brain DHS samples at scale, generating footprints—signatures of occupancy by DNA binding proteins—using the Wellington and HINT algorithms. We assembled motifs from JASPAR2016, HOCOMOCO, UniPROBE, and SwissRegulon, removing redundant motifs with Tomtom and intersecting our footprints with all possible overlapping motifs. This resulted in a total of 1,530motifs mapping to 1,515 different transcription factors.We developed and utilized Transcriptional Regulatory Network Analysis (TReNA), available as an R Bioconductor package. Gene regulatory regions considered in our model were obtained through Genehancer, thus enabling the inclusion of all known enhancer regions. TReNA utilizes an ensemble of machine learning techniques, including lassopv, square root lasso (flare) and randomForest to prioritize transcription factors based on the expression levels in RNA-seq for each target gene. The scores from the aforementioned techniques are scaled and normalized into a composite score, thereby ranking all associated transcription factors for each target gene. Results:We have identified transcriptional regulators for AD genes identified through GWAS. We have also identified putative targets for the AD-associated transcription factor MEF2C. We have identified multiple microglia-enriched transcription factors that regulate many differentially and co-expressed genes in AD, in particular, the AD-associated transcription factor SPI1. Conclusions:These resulting models can be applied to other datasets that generate lists of differentially or co-expressed genes as well as provide testable hypotheses for non-coding variants of interest. We are actively engaged in testing several hypotheses through experimental means and have made these TRNs publically available.

MODEL ORGANISM DEVELOPMENT AND EVALUATION FOR LATE-ONSET ALZHEIMER’S DISEASE: MODEL-AD

hippocampus and cortex in an APP/PSEN1 mouse. Methods:We utilize a cell-specific inducible system to express human TDP-43 and nuclear localization signal defective TDP-43DNLS in hippocampal and cortical neuronal populations using the Camk2a tetracycline transactivator (Camk2a-tTA) in an APP/PSEN1 background. The effect of TDP-43 on cognition and neurotoxicity was evaluated through novel object recognition, Y-maze, pathology, electron microscopy, immunofluorescence, primary cortical neuronal cultures, proteomics, and Western blotting. Results: We describe functional changes in short and long-term memory associated with TDP-43 expression. In addition, we describe pathological changes in AB plaques, tau aggregation, and phosphorylated TDP-43. Conclusions: Collectively, our data lead us to conclude that TDP-43 contributes to functional changes in memory, Ab plaque formation, tau aggregation, and accumulation of phosphorylated TDP-43. Our current data suggest that TDP-43 could be a putative target of therapeutic intervention in AD affecting both AB plaques and tauopathy.