Genes that code for proteins make up only about 2% of the human genome. Many researchers once dismissed the other 98% of the genome as “junk DNA,” but geneticists now know these noncoding regions help to regulate the activity of the 20,000 or so protein-coding genes identified.
A new study in Nature underscores just how important noncoding DNA can be for human development. The authors show that deletions in a noncoding region of DNA on chromosome 2 cause severe congenital limb abnormalities. This is the first time a human disease has been definitively linked to mutations in noncoding DNA, says lead author Stefan Mundlos, head of the development and disease research group at the Max Planck Institute for Molecular Genetics in Berlin, Germany.
“Interpreting the noncoding genome is still a big challenge just because we know so little about it,” he says. “We still haven’t managed to diagnose and discover mutations in the majority of patients with congenital malformations. People only usually look at the coding regions, the exome, and the noncoding is ignored.”
To make the discovery, the research team analyzed the DNA of three patients from Brazil and India, each born with a striking type of malformation. Their knees bend in the wrong direction and don’t orientate to the front, some fingers are fused, and nails grow from the other side of their fingers.
Comparative genomic hybridization showed each patient had homozygous deletions in a noncoding region close to a gene called engrailed-1 (EN1), which is known to be critical for dorsal–ventral (back–front) patterning in the limbs and has already been implicated in clinical limb-brain disorders. When the researchers used CRISPR–Cas gene editing to remove the corresponding noncoding DNA from mice, the animals showed the same kinds of developmental limb abnormalities seen in the human patients. This was associated with lower expression of the EN1 gene in the developing mouse limb bud. Expression of EN1 elsewhere was unaffected, suggesting the deleted sequence contained regulatory elements specific to EN1 expression in the limb.
To home in on this molecular mechanism, the scientists took a closer look at gene expression within the mouse embryo limb buds. They found the deleted region of DNA usually transcribed a string of RNA, which they called Maenli. When transcription of this lncRNA (long noncoding RNA, pronounced “link-RNA”) molecule was inactivated, EN1expression dropped by 90%.
Timothy Pullen, who works on lncRNA and its role in diabetes at King’s College London, says some cells express up to a thousand different lncRNA strands, but that it’s not clear what they do. “The big gap in long noncoding RNA research,” notes Pullen, who was not involved in the recent work, “is moving from just showing expression to showing that expression has some function.”
The Nature study is one of the first to make that connection, he says. The authors tweaked the mouse DNA further to show it was the transcriptional activity of the noncoding region—and not the presence of the specific Maenli snippet of lncDNA—that mattered for normal development. Different genetic expression at the same site was enough to activate the EN1 gene, although to a lesser extent than the original sequence. “It’s an RNA that works like an enhancer, but it is not an enhancer,” Mundlos says.
Igor Ulitsky, a lncRNA researcher at the Weizmann Institute of Science in Rehovot, Israel, says the study is a significant step forward. “They nicely show that even though these [noncoding] RNA transcripts don’t look exceptional they can be very important and very consequential, both on the expression of other genes and eventually on development and physiology.” Looking ahead, the study authors expect that whole-genome sequencing will reveal other mutations in noncoding regions likely to play a role in other rare genetic diseases.
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