ABSTRACT
The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts, separated by a hinge region. Using Hi-C in edited mouse cells with allelic deletions or inversions within the hinge, here we show that the conserved Dxz4 locus is necessary to maintain this bipartite structure. Dxz4 orientation controls the distribution of contacts on the Xi, as shown by a massive reversal in long-range contacts after Dxz4 inversion. Despite an increase in CTCF binding and chromatin accessibility on the Xi in Dxz4-edited cells, only minor changes in TAD structure and gene expression were detected, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of its bank of CTCF motifs, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.
Subject(s)
Alleles , CCCTC-Binding Factor/genetics , Epigenesis, Genetic , X Chromosome Inactivation , Amino Acid Motifs , Animals , CCCTC-Binding Factor/chemistry , Chromatin/chemistry , Chromatin/genetics , DNA Methylation , Gene Deletion , Gene Expression Regulation , Gene Silencing , In Situ Hybridization, Fluorescence , Mice , Mice, Inbred C57BL , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Protein Binding , X ChromosomeABSTRACT
Marfan syndrome (MFS) due to mutations in FBN1 is a known cause of thoracic aortic aneurysms and acute aortic dissections (TAAD) associated with pleiotropic manifestations. Genetic predisposition to TAAD can also be inherited in families in the absence of syndromic features, termed familial TAAD (FTAAD), and several causative genes have been identified to date. FBN1 mutations can also be identified in FTAAD families, but the frequency of these mutations has not been established. We performed exome sequencing of 183 FTAAD families and identified pathogenic FBN1 variants in five (2.7%) of these families. We also identified eight additional FBN1 rare variants that could not be unequivocally classified as disease-causing in six families. FBN1 sequencing should be considered in individuals with FTAAD even without significant systemic features of MFS.
Subject(s)
Aortic Aneurysm, Thoracic/genetics , Aortic Dissection/genetics , Fibrillin-1/genetics , Genetic Predisposition to Disease/genetics , Mutation , Adult , Aged , Aortic Dissection/pathology , Aortic Aneurysm, Thoracic/pathology , Exome/genetics , Family Health , Female , Humans , Male , Marfan Syndrome/genetics , Marfan Syndrome/pathology , Middle Aged , Pedigree , Sequence Analysis, DNA/methodsABSTRACT
BACKGROUND: Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterised by hypotonia, ataxia, cognitive impairment, abnormal eye movements, respiratory control disturbances and a distinctive mid-hindbrain malformation. JS demonstrates substantial phenotypic variability and genetic heterogeneity. This study provides a comprehensive view of the current genetic basis, phenotypic range and gene-phenotype associations in JS. METHODS: We sequenced 27 JS-associated genes in 440 affected individuals (375 families) from a cohort of 532 individuals (440 families) with JS, using molecular inversion probe-based targeted capture and next-generation sequencing. Variant pathogenicity was defined using the Combined Annotation Dependent Depletion algorithm with an optimised score cut-off. RESULTS: We identified presumed causal variants in 62% of pedigrees, including the first B9D2 mutations associated with JS. 253 different mutations in 23 genes highlight the extreme genetic heterogeneity of JS. Phenotypic analysis revealed that only 34% of individuals have a 'pure JS' phenotype. Retinal disease is present in 30% of individuals, renal disease in 25%, coloboma in 17%, polydactyly in 15%, liver fibrosis in 14% and encephalocele in 8%. Loss of CEP290 function is associated with retinal dystrophy, while loss of TMEM67 function is associated with liver fibrosis and coloboma, but we observe no clear-cut distinction between JS subtypes. CONCLUSIONS: This work illustrates how combining advanced sequencing techniques with phenotypic data addresses extreme genetic heterogeneity to provide diagnostic and carrier testing, guide medical monitoring for progressive complications, facilitate interpretation of genome-wide sequencing results in individuals with a variety of phenotypes and enable gene-specific treatments in the future.
Subject(s)
Cerebellum/abnormalities , Genetic Heterogeneity , Retina/abnormalities , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Cerebellum/pathology , Cohort Studies , DNA Mutational Analysis , Eye Abnormalities/genetics , Eye Abnormalities/pathology , Genetic Association Studies , Humans , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/pathology , Models, Theoretical , Pedigree , Retina/pathology , Sequence Analysis, DNAABSTRACT
Autism spectrum disorder (ASD) has a strong but complex genetic component. Here we report on the resequencing of 64 candidate neurodevelopmental disorder risk genes in 5,979 individuals: 3,486 probands and 2,493 unaffected siblings. We find a strong burden of de novo point mutations for these genes and specifically implicate nine genes. These include CHD2 and SYNGAP1, genes previously reported in related disorders, and novel genes TRIP12 and PAX5. We also show that mutation carriers generally have lower IQs and enrichment for seizures. These data begin to distinguish genetically distinct subtypes of autism important for aetiological classification and future therapeutics.
Subject(s)
Autistic Disorder/genetics , Genetic Predisposition to Disease , Base Sequence , Carrier Proteins/genetics , DNA-Binding Proteins/genetics , Family , Humans , Intelligence/genetics , Intelligence Tests , Mutation , PAX5 Transcription Factor/genetics , Risk , Sequence Analysis, DNA , Ubiquitin-Protein Ligases/genetics , ras GTPase-Activating Proteins/geneticsABSTRACT
The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.
Subject(s)
Disease , Genetic Predisposition to Disease/genetics , Genetic Variation/genetics , Guidelines as Topic , False Positive Reactions , Genes/genetics , Humans , Information Dissemination , Publishing , Reproducibility of Results , Research Design , Translational Research, Biomedical/standardsABSTRACT
We describe a method of genome-wide analysis of quantitative growth phenotypes using insertional mutagenesis and DNA microarrays. We applied the method to assess the fitness contributions of Escherichia coli gene domains under specific growth conditions. A transposon library was subjected to competitive growth selection in Luria-Bertani (LB) and in glucose minimal media. Transposon-containing genomic DNA fragments from the selected libraries were compared with the initial unselected transposon insertion library on DNA microarrays to identify insertions that affect fitness. Genes involved in the biosynthesis of nutrients not provided in the growth medium were found to be significantly enriched in the set of genes containing negatively selected insertions. The data also identify fitness contributions of several uncharacterized genes, including putative transcriptional regulators and enzymes. The applicability of this high-resolution array selection in other species is discussed.
Subject(s)
DNA Footprinting/methods , Escherichia coli/genetics , Mutagenesis, Insertional/methods , Oligonucleotide Array Sequence Analysis/methods , Culture Media , DNA Transposable Elements , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/physiology , Genome, Bacterial , Genomic Library , PhenotypeABSTRACT
We are in the enviable position of having two distinct drafts of the human genome sequence. Although gaps, errors, redundancy and incomplete annotation mean that individually each falls short of the ideal, many of these problems can be assessed by comparison. Here we present some comparative analyses of these drafts. We look at a number of features of the sequences, including sequence gaps, continuity, consistency between the two sequences and patterns of DNA-binding protein motifs.
Subject(s)
Genome, Human , Human Genome Project , Algorithms , Binding Sites , DNA/metabolism , DNA-Binding Proteins/metabolism , Databases, Factual , Humans , Private Sector , Public SectorABSTRACT
We report on the properties of a set of HIV-1 IIIB Env mutants carrying a linear gp41 epitope insertion (LLELDKWASL) in the V1, V2, V3 or V4 variable loop. Insertion of the epitope, which is defined by the HIV-1 neutralizing MAb 2F5, was well tolerated in the V1, V2 and V4 loops, as these mutants were properly expressed, retained reactivity to conformation-dependent monoclonal antibodies and exhibited patterns similar to the parental Env molecule. However, insertion of this epitope in the V3 loop was associated with drastically reduced protein expression. Relative to parental Env molecule, the V1, V2 and V4 insertion mutants demonstrated significantly increased binding to mAb 2F5 in vitro. To evaluate immunogenicity, mice and guinea pigs were immunized with plasmid expression vectors for the mutant proteins. For both mice and guinea pigs, all four mutants elicited anti-gp120 antibody responses. In mice the V1 and V3 insertion mutants, but neither the V2 or V4 insertion mutant nor the parental env, elicited significant titers against the epitope peptide, whereas in guinea pigs, V2 insertion mutant was most effective in eliciting anti-2F5 peptide antibody responses. While original V2 2F5 insertion mutant failed to elicit anti-2F5 peptide responses in mice, studies with 14 additional V2 insertion mutants revealed several insertion sites at which the epitope was able to induce epitope-specific antibody responses. This indicates that the precise position at which the epitope insertion takes place dictates the ability of the mutant to induce the epitope-specific antibody responses. When tested for virus neutralization activity, the guinea pig sera that contain high titers of anti-2F5 peptide antibody failed to enhance the virus neutralizing activity, suggesting that the configuration of 2F5 epitope plays a critical role in inducing neutralizing antibody responses. The results from this study may have potential implications with respect to modification of the HIV-1 Env molecule for the purpose of improving HIV-1 Env immunogenicity.
Subject(s)
AIDS Vaccines/genetics , AIDS Vaccines/immunology , Epitopes/genetics , Epitopes/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Viral/biosynthesis , Cell Line , Female , Guinea Pigs , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutagenesis, Insertional , Neutralization Tests , Vaccines, Synthetic/geneticsABSTRACT
We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse Chromosome (Chr) 11, occurring in the offspring of crosses between two commonly used Mus musculus-derived inbred strains, C57BL/6J and DBA/2J. In the surviving backcross progeny of reciprocal matings between (C57BL/6J x DBA/2J)F1 hybrids and the C57BL/6J parental strain, we observed the preferential appearance of C57BL/6J alleles along a region of Chr 11. The deviation from Mendelian predictions was observed only in female offspring from both reciprocal backcrosses, and not in males from either cross. The sex-specificity of the observed non-Mendelian inheritance points to an explanation based on embryonic or neonatal lethality. Our data add to previously obtained evidence for a Chr 11 locus or loci with sex-specific and allele-specific effects on viability.
Subject(s)
Chromosomes/genetics , Crosses, Genetic , Alleles , Animals , Female , Fetal Death/genetics , Genotype , Heterozygote , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Phenotype , Pregnancy , Sex CharacteristicsABSTRACT
C57Bl/6 mice reproducibly prefer to ingest more 10% ethanol in a two-bottle choice paradigm than do DBA/2J mice. In this paper we report the identification of two new sex-specific alcohol preference (Alcp) loci. Melo and associates (1996) identified two loci: Alcp1, a male-specific locus on Chromosome (Chr) 2, and Alcp2, a female- and cross-specific locus on Chr 11. We have additionally identified Alcp3, a male-specific locus on Chr 3, and Alcp4, a female-specific locus on Chr 1. We have also performed a statistical analysis to exclude the possibility of undiscovered major alcohol preference loci that are not sex-specific in our backcross paradigm. Our results indicate that alcohol preference in C57BL/6 mice, as measured in our backcross, is largely controlled in a sex-specific manner.
Subject(s)
Alcohol Drinking/genetics , Quantitative Trait, Heritable , Animals , Chromosome Mapping , Chromosomes/genetics , Crosses, Genetic , Epistasis, Genetic , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Sex FactorsABSTRACT
Mice from various inbred strains consume alcoholic beverages at highly reproducible and strain-specific levels. While most mice consume alcohol in moderate amounts, C57BL/6J animals exhibit sustained oral ingestion of high levels of alcohol in the presence of competing water and food. We now report a genetic investigation of this phenotype as one potential model for alcoholism. An intercross-backcross breeding protocol was used to identify two recessive alcohol preference quantitative trait loci (QTLs) that are both sex-restricted in expression. A comparison of our results with those of an earlier morphine preference study argues against the hypothesis of a single unified phenotype defined by a preference for all euphoria-producing drugs.