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SRX8178505: GSM4498653: mpimg_L19076-1_E9-5-FB-Rep1-cHiC; Mus musculus; OTHER
1 ILLUMINA (Illumina HiSeq 2500) run: 162.5M spots, 16.3G bases, 4.6Gb downloads

Submitted by: NCBI (GEO)
Study: Loss of Maenli lncRNA expression causes engrailed-1 dependent congenital limb malformations [Capture Hi-C]
show Abstracthide Abstract
Long non-coding RNAs (lncRNAs) can be important components in gene regulatory networks1, but we are only beginning to understand the nature and extent of their involvement in human Mendelian disease. Here we show that deletions of an unannotated lncRNA locus on human chromosome 2 cause a severe congenital limb malformation. Using exome sequencing and array CGH, we identified homozygous 27-63 kb deletions located 300 kb upstream of the engrailed-1 (EN1) gene in patients with a complex limb malformation, featuring mesomelic shortening, syndactyly, and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of limb-specific En1 expression and a double dorsal limb phenotype, recapitulating the human malformation. Genome-wide analysis in the developing mouse limb revealed the presence of a 4-exon long non-coding transcript within the deleted region, which we named Maenli (for Master activator of En1 in the limb). Functional dissection of the Maenli locus showed that limb-specific En1 expression depends on transcription of Maenli and its loss resulted in the double dorsal limb phenotype. Concomitant monoallelic inactivation of En1 and Maenli in double heterozygous mice did not rescue the limb phenotype, indicating that En1 activation in the limb requires the cis-acting regulatory element Maenli. Moreover, our results strongly suggest that En1 activation is dependent on Maenli transcription, but not on the Maenli RNA itself. Thus, Maenli expression in the limb acts in cis to promote En1 transcriptional activation; its loss results in congenital malformation of the limbs, a subset of the full En1 associated phenotype. Together, our findings provide evidence that mutations involving lncRNAs loci can result in human Mendelian disease. Overall design: We used exome sequencing and array CGH to analyze patients' samples, CRISPR experiments to investigate the effect of the potential pathogenic variants, genome wide analysis to identify a new lncRNA locus, and functional studies to characterize the effect of the knockout lncRNA locus.
Sample: mpimg_L19076-1_E9-5-FB-Rep1-cHiC
SAMN14733036 • SRS6538198 • All experiments • All runs
Organism: Mus musculus
Library:
Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: The capture HiC protocol was performed as decribed in (Formation of new chromatin domains determines pathogenecity of genomic duplications. Franke et al., Nature 2016). Snap frozen samples were first digested using DpnII, religated using the T4 DNA ligase (Thermo Fisher Scientific). The chimeric chromatin products were then decrosslinked overnight. RNase A treated chromatin was then precipiated.The precipiated chromatin was sheared using a Covaris sonicator (duty cycle: 10%; intensity: 5; cycles per burst: 200; time: 6 cycles of 60 s each; set mode: frequency sweeping; temperature: 4–7 °C). Adaptors were added to the sheared DNA and amplified according to the manufacturer's instructions for Illumina sequencing (Agilent). The library was hybridized to the custom-designed SureSelect beads and indexed for sequencing following the manufacturer's instructions (Agilent).
Experiment attributes:
GEO Accession: GSM4498653
Links:
Runs: 1 run, 162.5M spots, 16.3G bases, 4.6Gb
Run# of Spots# of BasesSizePublished
SRR11612240162,544,13416.3G4.6Gb2020-10-26

ID:
10662694

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