Non-coding genetic variants in human disease
Final Report Abstract
NGS technologies enable the simultaneous investigation of the entire genome. However, 40% of patients remain without molecular diagnosis despite that fact that on average ~80 de novo SNVs per patients are identified. The sheer number of these variants, overwhelmingly non-coding, make classical functional workup strategies impractical. In the first part of the project, we performed whole genome sequencing of 50 trios affected with congenital limb malformations, and followed this with functional characterization of all observed non-coding de novo SNVs via massively parallel reporter assays (MPRAs). All patients included were array CGH and exome negative. In total we identified 3,396 de novo mutations in the 50 patients. 5 de novo mutations were located in predicted enhancer regions based on epigenetics marks. For two of these predicted enhancers we could show positive in vivo enhancer activity in transgenic mouse reporter assays. Next, we used microarray-based DNA synthesis to create 230 bp oligonucleotides containing all 3,396 de novo non-coding variants and the corresponding wild-type sequences and perform lentivirus-based MPRAs in human chondrocyte cells and primary mouse limb bud cells. We experimentally measured the cis-regulatory activity of 3,396 de novo non-coding mutations in a single, quantitative experiment. We identified 48 variants that showed significant differential expression of the reporter gene. The positive candidates showed up to 5-fold enrichment for ENCODE TF binding motifs indicating that the mutations are likely to change TF binding and thereby contribute to disease. Our study provides a conceptual framework for the experimental assessment of the large number of de novo non-coding mutations from WGS studies. In the second part of the project, we wanted to study non-coding mutations and structural variants during embryogenesis. As a first step towards understanding pleiotropic developmental disorders at the organismal scale, we created a single cell atlas of the embryonic development of wild-type mice. We studied the transcriptional dynamics of mouse development during organogenesis at single cell resolution. With an improved single cell combinatorial indexing-based protocol ('sci-RNA-seq3'), we profiled over 2 million single cells derived from 61 mouse embryos staged between 9.5 and 13.5 days of gestation (E9.5 to E13.5; 10-15 replicates per time-point) in one, multiplex experiment. We identify major transcriptional lineages as well as hundreds of expanding, contracting and transient cell types, many of which are only detected because of the depth of cellular coverage obtained here. We explore the dynamics of gene expression within cell types over time, and infer pseudo-temporal trajectories of mouse limb and muscle development, including examples of distinct trajectories to the same endpoint. These data comprise a foundational resource for mammalian developmental biology. These data are of enormous interest for human genetics as genotype-phenotype correlations are extremely difficult to understand since the severity of genetic disorders can differ even in individuals with mutations in the same gene.
Publications
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(2018) Structural variation in the 3D genome. Nature reviews. Genetics 19 (7) 453–467
Spielmann, Malte; Lupiáñez, Darío G.; Mundlos, Stefan
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(2019) The single-cell transcriptional landscape of mammalian organogenesis. Nature 566 (7745) 496–502
Cao, Junyue; Spielmann, Malte; Qiu, Xiaojie; Huang, Xingfan; Ibrahim, Daniel M.; Hill, Andrew J.; Zhang, Fan; Mundlos, Stefan; Christiansen, Lena; Steemers, Frank J.; Trapnell, Cole; Shendure, Jay
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Mutations in MYO1H cause a recessive form of central hypoventilation with autonomic dysfunction J Med Genet. 2017 Nov;54(11):754-761
Spielmann M, Hernandez-Miranda LH, Ceccherini I, Weese-Mayer DE, Kragesteen BK, Harabula I, Krawitz P, Birchmeier C, Leonard N, Mundlos S
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Non-coding copy number variations are associated with congenital limb malformation Genet Med. Epub 2017 Oct 12
Flöttmann R, Kragesteen BK, Geuer S, Socha M, Allou L, Sowińska-Seidler A, Bosquillon de Jarcy L, Wagner J, Jamsheer A, Oehl-Jaschkowitz B, Wittler L, de Silva D, Kurth I, Maya I, Santos-Simarro F, Hülsemann W, Klopocki E, Mountford R, Fryer A, Borck G, Horn D, Lapunzina P, Wilson M, Mascrez B, Duboule D, Mundlos S, Spielmann M
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Dynamic 3D chromatin architecture contributes to enhancer specificity and limb morphogenesis. Nat Genet. 2018 Oct;50:1463-1473
Kragesteen BK, Spielmann M, Paliou C, Heinrich V, Schöpflin R, Esposito A, Annunziatella C, Bianco S, Chiariello AM, Jerković I, Harabula I, Guckelberger P, Pechstein M, Wittler L, Chan WL, Franke M, Lupiáñez DG, Kraft K, Timmermann B, Vingron M, Visel A, Nicodemi M, Mundlos S, Andrey G
