Malte Spielmann

Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

Formation of new chromatin domains determines pathogenicity of genomic duplications

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

Effective diagnosis of genetic disease by computational phenotype analysis of the disease-associated genome

Patients with genetic disease of unknown causes can be rapidly diagnosed by bioinformatic analysis of disease-associated DNA sequences and phenotype. Efficient Diagnosis of Genetic Disease We know which genes are mutated in almost 3000 inherited human diseases and have good descriptions of how these mutations affect the human phenotype. Now, Zemojtel et al. have coupled this knowledge with rapid sequencing of these genes in a group of 40 patients with undiagnosed genetic diseases. Bioinformatic matching of the patients’ clinical characteristics and their disease gene sequences to databases of current genetic and phenotype knowledge enabled the authors to successfully diagnose almost 30% of the patients. The process required only about 2 hours of a geneticists’ time. Zemojtel et al. have made their tools available to the community, enabling a fast straightforward process by which clinicians and patients can easily identify the genetic basis of inherited disease in certain people. Less than half of patients with suspected genetic disease receive a molecular diagnosis. We have therefore integrated next-generation sequencing (NGS), bioinformatics, and clinical data into an effective diagnostic workflow. We used variants in the 2741 established Mendelian disease genes [the disease-associated genome (DAG)] to develop a targeted enrichment DAG panel (7.1 Mb), which achieves a coverage of 20-fold or better for 98% of bases. Furthermore, we established a computational method [Phenotypic Interpretation of eXomes (PhenIX)] that evaluated and ranked variants based on pathogenicity and semantic similarity of patients’ phenotype described by Human Phenotype Ontology (HPO) terms to those of 3991 Mendelian diseases. In computer simulations, ranking genes based on the variant score put the true gene in first place less than 5% of the time; PhenIX placed the correct gene in first place more than 86% of the time. In a retrospective test of PhenIX on 52 patients with previously identified mutations and known diagnoses, the correct gene achieved a mean rank of 2.1. In a prospective study on 40 individuals without a diagnosis, PhenIX analysis enabled a diagnosis in 11 cases (28%, at a mean rank of 2.4). Thus, the NGS of the DAG followed by phenotype-driven bioinformatic analysis allows quick and effective differential diagnostics in medical genetics.

Breaking TADs: How Alterations of Chromatin Domains Result in Disease

Spatial organization is an inherent property of the vertebrate genome to accommodate the roughly 2m of DNA in the nucleus of a cell. In this nonrandom organization, topologically associating domains (TADs) emerge as a fundamental structural unit that is thought to guide regulatory elements to their cognate promoters. In this review we summarize the most recent findings about TADs and the boundary regions separating them. We discuss how the disruption of these structures by genomic rearrangements can result in gene misexpression and disease.

Comparison of Exome and Genome Sequencing Technologies for the Complete Capture of Protein-Coding Regions

For next‐generation sequencing technologies, sufficient base‐pair coverage is the foremost requirement for the reliable detection of genomic variants. We investigated whether whole‐genome sequencing (WGS) platforms offer improved coverage of coding regions compared with whole‐exome sequencing (WES) platforms, and compared single‐base coverage for a large set of exome and genome samples. We find that WES platforms have improved considerably in the last years, but at comparable sequencing depth, WGS outperforms WES in terms of covered coding regions. At higher sequencing depth (95x–160x), WES successfully captures 95% of the coding regions with a minimal coverage of 20x, compared with 98% for WGS at 87‐fold coverage. Three different assessments of sequence coverage bias showed consistent biases for WES but not for WGS. We found no clear differences for the technologies concerning their ability to achieve complete coverage of 2,759 clinically relevant genes. We show that WES performs comparable to WGS in terms of covered bases if sequenced at two to three times higher coverage. This does, however, go at the cost of substantially more sequencing biases in WES approaches. Our findings will guide laboratories to make an informed decision on which sequencing platform and coverage to choose.

Deletions of chromosomal regulatory boundaries are associated with congenital disease

BackgroundRecent data from genome-wide chromosome conformation capture analysis indicate that the human genome is divided into conserved megabase-sized self-interacting regions called topological domains. These topological domains form the regulatory backbone of the genome and are separated by regulatory boundary elements or barriers. Copy-number variations can potentially alter the topological domain architecture by deleting or duplicating the barriers and thereby allowing enhancers from neighboring domains to ectopically activate genes causing misexpression and disease, a mutational mechanism that has recently been termed enhancer adoption.ResultsWe use the Human Phenotype Ontology database to relate the phenotypes of 922 deletion cases recorded in the DECIPHER database to monogenic diseases associated with genes in or adjacent to the deletions. We identify combinations of tissue-specific enhancers and genes adjacent to the deletion and associated with phenotypes in the corresponding tissue, whereby the phenotype matched that observed in the deletion. We compare this computationally with a gene-dosage pathomechanism that attempts to explain the deletion phenotype based on haploinsufficiency of genes located within the deletions. Up to 11.8% of the deletions could be best explained by enhancer adoption or a combination of enhancer adoption and gene-dosage effects.ConclusionsOur results suggest that enhancer adoption caused by deletions of regulatory boundaries may contribute to a substantial minority of copy-number variation phenotypes and should thus be taken into account in their medical interpretation.

A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD).

Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (∼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

Structural variations, the regulatory landscape of the genome and their alteration in human disease.

High-throughput genomic technologies are revolutionizing human genetics. So far the focus has been on the 1.5% of the genome, which is coding, in spite of the fact that the great majority of genomic variants fall outside the coding regions. Recent efforts to annotate the non-coding sequence show that over 80% of the genome is biochemically active. The genome is divided into regulatory domains consisting of sequence regions that enhance and/or silence the expression of nearby genes and are, in some cases, separated by boundaries with insulator activity. In this paper, we review the recent advances in the identification of variations that influence gene regulation and their consequences for human disease. We hypothesize that structural variations outside of the coding genome can interfere with normal gene regulation by disrupting the regulatory landscape. Therefore, the regulatory landscape of the genome has also to be taken into consideration when investigating the pathology of human disease.

Homozygous deletion of chromosome 15q13.3 including CHRNA7 causes severe mental retardation, seizures, muscular hypotonia, and the loss of KLF13 and TRPM1 potentially cause macrocytosis and congenital retinal dysfunction in siblings.

The heterozygous 15q13.3 microdeletion syndrome (MIM #612001) was first described by Sharp et al. in 2008. So far four patients with 15q13.3 homozygous or compound heterozygous microdeletions have been identified. Here we report a non-consanguineous family with two affected siblings carrying a homozygous microdeletion of ∼1.5 Mb at the 15q13.3 locus. They presented with congenital retinal dysfunction, refractory epilepsy, encephalopathy, mental retardation, repetitive hand movements, severe muscular hypotonia and macrocytosis. Dysmorphic facial features are synophrys and bilateral proptosis. The siblings carry a homozygous microdeletion at 15q13.3 of ∼1.5 Mb including the genes ARHGAP11B, MTMR15, MTMR10, TRPM1, KLF13, OTUD7A, and CHRNA7. The absence of CHRNA7 has been suggested as a cause of refractory seizures. According to knock-out experiments the deletion of KLF13 could be an explanation for macrocytosis. The homozygous loss of TRPM1 could be a possible explanation for congenital retinal dysfunction.

CNVs of noncoding cis-regulatory elements in human disease

Genomic rearrangements and copy-number variations (CNVs) are structural aberrations of the human genome which contribute to phenotypic variation as well as human disease. By now it is well accepted that structural aberrations affecting coding regions can have pathogenic effects, however, noncoding variants have only recently come into focus as disease-associated variants. The phenotypes associated with alterations in noncoding regions with regulatory potential can be striking and at the same time confined to a certain tissue/organ. Future studies will elucidate the frequency of these changes which are expected to be higher among conditions that are due to disturbance of complex developmental processes. Integrating these data with the recently published data from the ENCODE project will broaden our view of genes and their regulation and contribute to our understanding of pathomechanism underlying human disease. In this article, we review the recent advances in the identification of genomic rearrangements and CNVs in noncoding regions of the genome and their consequences for human disease.