Showing papers by "Stylianos E. Antonarakis published in 2017"

Down syndrome and the complexity of genome dosage imbalance

Abstract: Down syndrome (also known as trisomy 21) is the model human phenotype for all genomic gain dosage imbalances, including microduplications. The functional genomic exploration of the post-sequencing years of chromosome 21, and the generation of numerous cellular and mouse models, have provided an unprecedented opportunity to decipher the molecular consequences of genome dosage imbalance. Studies of Down syndrome could provide knowledge far beyond the well-known characteristics of intellectual disability and dysmorphic features, as several other important features, including congenital heart defects, early ageing, Alzheimer disease and childhood leukaemia, are also part of the Down syndrome phenotypic spectrum. The elucidation of the molecular mechanisms that cause or modify the risk for different Down syndrome phenotypes could lead to the introduction of previously unimaginable therapeutic options.

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

Abstract: By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.

Systematic proteome and proteostasis profiling in human Trisomy 21 fibroblast cells

Abstract: Down syndrome (DS) is mostly caused by a trisomy of the entire Chromosome 21 (Trisomy 21, T21). Here, we use SWATH mass spectrometry to quantify protein abundance and protein turnover in fibroblasts from a monozygotic twin pair discordant for T21, and to profile protein expression in 11 unrelated DS individuals and matched controls. The integration of the steady-state and turnover proteomic data indicates that protein-specific degradation of members of stoichiometric complexes is a major determinant of T21 gene dosage outcome, both within and between individuals. This effect is not apparent from genomic and transcriptomic data. The data also reveal that T21 results in extensive proteome remodeling, affecting proteins encoded by all chromosomes. Finally, we find broad, organelle-specific post-transcriptional effects such as significant downregulation of the mitochondrial proteome contributing to T21 hallmarks. Overall, we provide a valuable proteomic resource to understand the origin of DS phenotypic manifestations.

Detection of Imprinted Genes by Single-Cell Allele-Specific Gene Expression

Abstract: Genomic imprinting results in parental-specific gene expression. Imprinted genes are involved in the etiology of rare syndromes and have been associated with common diseases such as diabetes and cancer. Standard RNA bulk cell sequencing applied to whole-tissue samples has been used to detect imprinted genes in human and mouse models. However, lowly expressed genes cannot be detected by using RNA bulk approaches. Here, we report an original and robust method that combines single-cell RNA-seq and whole-genome sequencing into an optimized statistical framework to analyze genomic imprinting in specific cell types and in different individuals. Using samples from the probands of 2 family trios and 3 unrelated individuals, 1,084 individual primary fibroblasts were RNA sequenced and more than 700,000 informative heterozygous single-nucleotide variations (SNVs) were genotyped. The allele-specific coverage per gene of each SNV in each single cell was used to fit a beta-binomial distribution to model the likelihood of a gene being expressed from one and the same allele. Genes presenting a significant aggregate allelic ratio (between 0.9 and 1) were retained to identify of the allelic parent of origin. Our approach allowed us to validate the imprinting status of all of the known imprinted genes expressed in fibroblasts and the discovery of nine putative imprinted genes, thereby demonstrating the advantages of single-cell over bulk RNA-seq to identify imprinted genes. The proposed single-cell methodology is a powerful tool for establishing a cell type-specific map of genomic imprinting.

The effect of genetic variation on promoter usage and enhancer activity.

Abstract: The identification of genetic variants affecting gene expression, namely expression quantitative trait loci (eQTLs), has contributed to the understanding of mechanisms underlying human traits and diseases. The majority of these variants map in non-coding regulatory regions of the genome and their identification remains challenging. Here, we use natural genetic variation and CAGE transcriptomes from 154 EBV-transformed lymphoblastoid cell lines, derived from unrelated individuals, to map 5376 and 110 regulatory variants associated with promoter usage (puQTLs) and enhancer activity (eaQTLs), respectively. We characterize five categories of genes associated with puQTLs, distinguishing single from multi-promoter genes. Among multi-promoter genes, we find puQTL effects either specific to a single promoter or to multiple promoters with variable effect orientations. Regulatory variants associated with opposite effects on different mRNA isoforms suggest compensatory mechanisms occurring between alternative promoters. Our analyses identify differential promoter usage and modulation of enhancer activity as molecular mechanisms underlying eQTLs related to regulatory elements.

MBV: a method to solve sample mislabeling and detect technical bias in large combined genotype and sequencing assay datasets

Abstract: Motivation: Large genomic datasets combining genotype and sequence data, such as for expression quantitative trait loci (eQTL) detection, require perfect matching between both data types. Results: We described here MBV (Match BAM to VCF); a method to quickly solve sample mislabeling and detect cross-sample contamination and PCR amplification bias. Availability and Implementation: MBV is implemented in C ++ as an independent component of the QTLtools software package, the binary and source codes are freely available at https://qtltools.github.io/qtltools/. Contact: olivier.delaneau@unige.ch or emmanouil.dermitzakis@unige.ch Supplementary information: Supplementary data are available at Bioinformatics online.

Genome-Wide Association Study to Find Modifiers for Tetralogy of Fallot in the 22q11.2 Deletion Syndrome Identifies Variants in the GPR98 Locus on 5q14.3

Abstract: Background— The 22q11.2 deletion syndrome (22q11.2DS; DiGeorge syndrome/velocardiofacial syndrome) occurs in 1 of 4000 live births, and 60% to 70% of affected individuals have congenital heart disease, ranging from mild to severe. In our cohort of 1472 subjects with 22q11.2DS, a total of 62% (n=906) have congenital heart disease and 36% (n=326) of these have tetralogy of Fallot (TOF), comprising the largest subset of severe congenital heart disease in the cohort. Methods and Results— To identify common genetic variants associated with TOF in individuals with 22q11.2DS, we performed a genome-wide association study using Affymetrix 6.0 array and imputed genotype data. In our cohort, TOF was significantly associated with a genotyped single-nucleotide polymorphism (rs12519770, P =2.98×10 − 8 ) in an intron of the adhesion GPR98 (G-protein–coupled receptor V1) gene on chromosome 5q14.3. There was also suggestive evidence of association between TOF and several additional single-nucleotide polymorphisms in this region. Some genome-wide significant loci in introns or noncoding regions could affect regulation of genes nearby or at a distance. On the basis of this possibility, we examined existing Hi-C chromatin conformation data to identify genes that might be under shared transcriptional regulation within the region on 5q14.3. There are 6 genes in a topologically associated domain of chromatin with GPR98 , including MEF2C (Myocyte-specific enhancer factor 2C). MEF2C is the only gene that is known to affect heart development in mammals and might be of interest with respect to 22q11.2DS. Conclusions— In conclusion, common variants may contribute to TOF in 22q11.2DS and may function in cardiac outflow tract development.

The genomic landscape of human cellular circadian variation points to a novel role for the signalosome

Abstract: The importance of natural gene expression variation for human behavior is undisputed, but its impact on circadian physiology remains mostly unexplored. Using umbilical cord fibroblasts, we have determined by genome-wide association how common genetic variation impacts upon cellular circadian function. Gene set enrichment points to differences in protein catabolism as one major source of clock variation in humans. The two most significant alleles regulated expression of COPS7B, a subunit of the COP9 signalosome. We further show that the signalosome complex is imported into the nucleus in timed fashion to stabilize the essential circadian protein BMAL1, a novel mechanism to oppose its proteasome-mediated degradation. Thus, circadian clock properties depend in part upon a genetically-encoded competition between stabilizing and destabilizing forces, and genetic alterations in these mechanisms provide one explanation for human chronotype.

Congenital Neuronal Ceroid Lipofuscinosis with a Novel CTSD Gene Mutation: A Rare Cause of Neonatal-Onset Neurodegenerative Disorder.

Abstract: Neuronal ceroid lipofuscinoses represent a heterogeneous group of early onset neurodegenerative disorders that are characterized by progressive cognitive and motor function decline, visual loss, and epilepsy. The age of onset has been historically used for the phenotypic classification of this group of disorders, but their molecular genetic delineation has now enabled a better characterization, demonstrating significant genetic heterogeneity even among individuals with a similar phenotype. The rare Congenital Neuronal Ceroid Lipofuscinosis (CLN10) caused by mutations in the CTSD gene encoding for cathepsin D is associated with a dramatic presentation with onset before or around birth. We report on a female born to consanguineous parents who presented at birth with severe neonatal encephalopathy with massive cerebral and cerebellar shrinking on magnetic resonance imaging. Whole exome sequencing with targeted bioinformatic analysis of a panel of genes associated with prenatal/perinatal onset of neurodegenerative disease was performed and revealed the presence of a novel homozygous in-frame deletion in CTSD. Additional functional studies further confirmed the pathogenic character of this variant and established the diagnosis of CLN10 in the patient.

Intra- and inter-chromosomal chromatin interactions mediate genetic effects on regulatory networks

Abstract: Genome-wide studies on the genetic basis of gene expression and the structural properties of chromatin have considerably advanced our understanding of the function of the human genome. However, it remains unclear how structure relates to function and, in this work, we aim at bridging both by assembling a dataset that combines the activity of regulatory elements (e.g. enhancers and promoters), expression of genes and genetic variations of 317 individuals and across two cell types. We show that the regulatory activity is structured within 12,583 Cis Regulatory Domains (CRDs) that are cell type specific and highly reflective of the local (i.e. Topologically Associating Domains) and global (i.e. A/B nuclear compartments) nuclear organization of the chromatin. These CRDs essentially delimit the sets of active regulatory elements involved in the transcription of most genes, thereby capturing complex regulatory networks in which the effects of regulatory variants are propagated and combined to finally mediate expression Quantitative Trait Loci. Overall, our analysis reveals the complexity and specificity of cis and trans regulatory networks and their perturbation by genetic variation.

Germline PMS2 and somatic POLE exonuclease mutations cause hypermutability of the leading DNA strand in biallelic mismatch repair deficiency syndrome brain tumours.

Abstract: Biallelic mismatch repair deficiency (bMMRD) in tumours is frequently associated with somatic mutations in the exonuclease domains of DNA polymerases POLE or POLD1, and results in a characteristic mutational profile. In this article, we describe the genetic basis of ultramutated high-grade brain tumours in the context of bMMRD. We performed exome sequencing of two second-cousin patients from a large consanguineous family of Indian origin with early onset of high-grade glioblastoma and astrocytoma. We identified a germline homozygous nonsense variant, p.R802*, in the PMS2 gene. Additionally, by genome sequencing of these tumours, we found extremely high somatic mutation rates (237/Mb and 123/Mb), as well as somatic mutations in the proofreading domain of POLE polymerase (p.P436H and p.L424V), which replicates the leading DNA strand. Most interestingly, we found, in both cancers, that the vast majority of mutations were consistent with the signature of POLE exo- , i.e. an abundance of C>A and C>T mutations, particularly in special contexts, on the leading strand. We showed that the fraction of mutations under positive selection among mutations in tumour suppressor genes is more than two-fold lower in ultramutated tumours than in other glioblastomas. Genetic analyses enabled the diagnosis of the two consanguineous childhood brain tumours as being due to a combination of PMS2 germline and POLE somatic variants, and confirmed them as bMMRD/POLE exo- disorders. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

No evidence for the presence of genetic variants predisposing to psychotic disorders on the non-deleted 22q11.2 allele of VCFS patients.

Abstract: The velo-cardio-facial syndrome (VCFS) is caused by hemizygous deletions on chromosome 22q11.2. The VCFS phenotype is complex and characterized by frequent occurrence of neuropsychiatric symptoms with up to 25-30% of cases suffering from psychotic disorders compared with only ~1% in the general population (odds ratio≈20-25). This makes the 22q11.2 deletion one of the most prominent risk factors for schizophrenia. However, its penetrance for neuropsychiatric phenotypes is incomplete suggesting that additional risk factors are required for disease development. These additional risk factors could lie anywhere on the genome, but by reducing the normal diploid to a haploid state, the 22q11.2 deletion could result in the unmasking of otherwise recessive alleles or functional variants on the non-deleted 22q11.2 allele. To test this hypothesis, we captured and sequenced the whole 22q11.2 non-deleted region in 88 VCFS patients with (n=40) and without (n=48) psychotic disorders to identify genetic variation that could increase the risk for schizophrenia. Single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variants were called and their distributions were compared between the two diagnostic groups using variant-, gene- and region-based association tests. None of these tests resulted in statistical evidence for the existence of a genetic variation in the non-deleted allele that would increase schizophrenia risk in VCFS patients. Power analysis showed that our study was able to achieve >80% statistical power to detect association of a risk variant with an odd ratio of ⩾22. However, it is certainly under-powered to detect risk variant of smaller effect sizes. Our study did not provide evidence that genetic variants of very large effect size located on the non-deleted 22q1.2 allele in VCFS patients increase the risk for developing psychotic disorders. Variants with smaller effects may be located in the remaining 22q11.2 allele and elsewhere in the genome. Therefore, whole exome or even genome sequencing for larger sample size would appear to be the next logical steps in the search for the genetic modifiers of the 22q11.2-deletion neuropsychiatric phenotype.

SERPINI1 pathogenic variants: An emerging cause of childhood-onset progressive myoclonic epilepsy.

Abstract: Progressive myoclonic epilepsies are rare neurodegenerative diseases with a wide spectrum of clinical presentations and genetic heterogeneity that render their diagnosis perplexing. Discovering new imputable genes has been an ongoing process in recent years. We present two pediatric cases of progressive myoclonic epilepsy with SERPINI1 pathogenic variants that lead to a severe presentation; we highlight the importance of including this gene, previously known as causing an adult-onset dementia-epilepsy syndrome, in the genetic work-up of childhood-onset progressive myoclonic epilepsies.

Deciphering cell lineage specification during male sex determination with single-cell RNA sequencing

Abstract: The gonad is a unique biological system for studying cell fate decisions. However, major questions remain regarding the identity of somatic progenitor cells and the transcriptional events driving cell differentiation. Using time course single cell RNA sequencing on XY mouse gonads during sex determination, we identified a single population of somatic progenitor cells prior sex determination. A subset of these progenitors differentiate into Sertoli cells, a process characterized by a highly dynamic genetic program consisting of sequential waves of gene expression. Another subset of multipotent cells maintains their progenitor state but undergo significant transcriptional changes that restrict their competence towards a steroidogenic fate required for the differentiation of fetal Leydig cells. These results question the dogma of the existence of two distinct somatic cell lineages at the onset of sex determination and propose a new model of lineage specification from a unique progenitor cell population.

Viva Europa, a Land of Excellence in Research and Innovation for Health and Wellbeing

Abstract: Charles Auffraya; Michael Sagnerb; Sonia Abdelhakc; Ian Adcockd; Alvar Agustie; Margarida Amaralf; Stylianos Antonarakisg; Ross Arenah; Françoise Argouli; Rudi Ballingj; Albert-Laszlo Barabasik; Jacques Beckmannl; Anders Bjartellm; Niklas Blombergn; Thomas Bourgerono; Bertrand Boutronp; Samir Brahmachariq; Christian Bréchotr; Christopher Brightlings; Marta Cascantet; Alfredo Cesariou; Dominique Charronv; Sai-Juan Chenw; Zhu Chenx; Fan Chungy; Karine Clémentz; Ana Conesaaa; Alain Cozzoneab; Menno de Jongac; Jean-François Deleuzead; Jacques Demotesae; Alberto di Meglioaf; Ratko Djukanovicag; Ugur Dogrusozah; Elissa Epelai; Alain Fischeraj; Andrea Gelemanovicak; Carole Gobleal; Takashi Gojoboriam; Michel Goldmanan; Herman Goossensao; François Grosap; Yi-Ke Guoaq; Pierre Hainautar; David Harrisonas; Hans Hoffmannat; Leroy Hoodau; Peter Hunterav; Yves Jacobaw; Hiroaki Kitanoax; Ursula Klingmülleray; Bartha Knoppersaz; Walter Kolchba; Marion Koopmansbb; Doron Lancetbc; Martine Lavillebd; Jean-Marie Lehnbe; Francis Lévibf; Andrey Lisistsabg; Vincent Lotteaubh; Antoine Magnanbi; Bongani Mayosibj; Andres Metspalubk; Yves Moreaubl; James N’Dowbm; Laurent Nicodbn; Denis Noblebo; Maria Manuela Nogueirabp; Anna Norrby-Teglundbq; Laurent Nottalebr; Peter Openshawbs; Mehmet Oztürkbt; Susanna Palkonenbu; Silvio Parodibv; Johann Pelletbw; Ozren Polasekbx; Nathan Priceby; Christian Pristipinobz; Timothy Radstakeca; Martine Raescb; Josep Rocacc; Damjana Rozmancd; Philippe Sabatierce; Shlomo Sassoncf; Bernd Schmeckcg; Ismaïl Serageldinch; Anita Simondsci; Bento Soarescj; Peter Sterkck; Giulio Superti-Furgacl; David Supplecm; Jesper Tegnercn; Mathias Uhlenco; Sylvie van der Werfcp; Pablo Villosladacq; Manlio Vinciguerracr; Vitaly Volpertcs; Steve Webbct; Emiel Wouterscu; Ferran Sanzcv; Francisco Nobregacw

Genomic databases: A WHO affair.

Abstract: The use of the genome for diagnostic purposes is a routine practice for disorders that stem from a single gene. Yet only 3927 protein-coding genes—0.3% of the human genome—have been linked to such disorders ([ 1 ][1]). Even within this fraction of the genome, the majority of the rare variants

Improving Leukemic CD34+/CD38- Blasts Characterization With Single-Cell Transcriptome Sequencing

Abstract: Acute myeloid leukemia (AML) is a particularly aggressive blood cancer that is difficult to treat because of the incomplete eradication of rare blast cells that possess self-renewal and leukemia-initiating properties To characterize resistant blasts, we analyzed for the first time the transcriptomes of individual CD34+/CD38- blasts by single-cell mRNA sequencing of 359 CD33+/CD34+/CD38-/+ sorted cells from two patients with AML and four unaffected individuals We demonstrated that the captured blasts possess the transcriptomic hallmarks of self-renewal and leukemia-initiating ability The effects of somatic mutations on the cancer cells are visible at the transcriptional level, and the cellular signaling pathway activity of the blasts is altered, revealing disease-associated gene networks We also identified a core set of transcription factors that were co-activated in blasts, which suggests a joint transcription program among blasts Finally, we revealed that leukemogenesis and putative prognostic gene-expression signatures are present at diagnosis in leukemic CD33+/CD34+/CD38- cells and can be detected using a single-cell RNA sequencing approach