The ability of different genetic variants – changes to one or more building blocks of DNA – to cause disease, and to what extent, has historically been opaque. 

Geneticist and Crick group leader Greg Findlay has pioneered a new method in the hope of changing this. Called ‘saturation genome editing’, the new technique involves mapping every single variant in a given gene to work out what it does and pinpoint which changes are responsible for specific disorders.

Whilst Greg was refining these experiments, Nicky Whiffin, associate professor at the University of Oxford, had identified that mutations in a tiny gene were behind a rare inherited neurodevelopmental disorder, known as ReNU syndrome, which impacts brain function, development and motor skills. Children develop this syndrome if a single copy of the RNU4-2 gene is mutated in a specific way.

Nicky initially found that several distinct mutations in a critical region of the gene caused the condition, and she was keen to understand if some of these genetic variants led to more severe disease.

A small gene with big impact

Unlike most genes implicated in disease, RNU4-2 does not encode a protein. Instead, it produces an RNA component of the ‘spliceosome’, the molecular machinery that processes RNA before it is translated into proteins.

Nicky’s work was the first time a non-coding gene had been found to have such a significant role in any rare disorder. But some questions remained: only about twenty mutations had been linked to ReNU syndrome, despite many other variants occurring in people without the condition.

“We started working with Nicky to use saturation genome editing to identify other variants that could be pathogenic,” says Greg. “This was the first time we’d used the method on a non-coding gene, so we weren’t sure if it was going to work.”

Joachim De Jonghe, a former postdoc in Greg’s team, had the mammoth task of testing all the single-nucleotide variants (changes to just one DNA base at a time) in RNU4-2, described in a study published in Nature. 

“With a non-coding gene, there isn’t a strict DNA code to protein relationship; their functions are much less defined and studied,” says Joachim. “So, it is much harder to use current predictive tools to understand if a mutation has a detrimental effect.”

Joachim had to make some small but very important modifications to the method, and managed to install over 500 different RNU4-2 variants in human cells.

“We gave each variant a score, which reflects how harmful it was to the cells,” Joachim explains. “Strikingly, these scores correlated well with disease severity observed in people with ReNU syndrome.”

Beyond confirming these known variants, Joachim believes the results will help identify new cases of ReNU syndrome caused by newly identified variants.

HAP1 cells (top) edited to contain different genetic variants, which are represented by the different colours. Function scores are plotted for each of the 539 variants (bottom): the lower the variant in the plot, the more harmful it is.

Reading between the lines

While analysing the data, Joachim and Greg identified a second, unexpected set of mutations that significantly impaired gene function, but were not in the same region of the gene linked to ReNU syndrome. 

Through collaboration with Nicky’s group at the University of Oxford and clinical teams working across the UK, Australia, France, Germany and the US, they demonstrated that these mutations cause a distinct neurodevelopmental disorder inherited in a recessive manner, meaning individuals need two mutations to be affected by the disorder, inheriting one from each parent.

“There was another disease hiding in the genome,” says Greg. “This shows the power of systematically testing every possible mutation in a gene.”

A serendipitous discovery

Now that the second disease had been identified, Nicky and her clinical collaborators were able to diagnose 38 individuals with recessive ReNU syndrome, reported in a study published in Nature Genetics today. Whilst sharing some features with the dominant ReNU syndrome caused by a single mutation, primarily severe developmental delay, the recessive syndrome was found to impact the white matter of the brain differently, a feature distinguishable by MRI. The teams also showed that the recessive mutations cause disease through a different mechanism. 

“Knowing exactly which DNA changes impair the function of the gene is a critical clinical tool,” says Nicky. “These studies not only improve our ability to diagnose patients, but also reveal entirely new biology that could be useful when designing treatments.” 

For Greg, the work stresses that experiments in the lab can have a profound effect on medicine. “So many people with rare disease remain undiagnosed on a genetic level. This work has strongly motivated us to do more experiments like this, to reveal new regions of the genome that cause rare disease,” he concludes. 

Oliver's story

Oliver Stuart is 15 and has the recessive form of ReNU syndrome. His mother Louise Bamford says that the long-awaited answer has been life-changing:

“From a very young age, Oliver showed signs of some kind of intellectual disability and physical disability and we went to a paediatrician who diagnosed him with Global Developmental Delay (GDD). We were then put in contact with Genomics England’s 100,000 Genomes Project and have waited years and years for an answer. So to get that phone call last year was an enormous surprise and huge relief.

“We’ve always known that there was something more with Oliver and that it wasn’t just global development or autism. And now having that answer has opened the doors to learning a lot about it. There will now be more information coming through and we can help other families who are yet to be diagnosed. We’ve joined the groups on Facebook already and it is so nice to be part of something and that we’ve now all got the answers that we’ve been searching for, for a long time.”

This research was made possible using data from the 100,000 Genomes Project. 

Adapted from an article from the University of Oxford, published on CrickNet by Clare Green