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Genetic Linkage

CRISPR Gene Editing of Neurons in Prader-Willi Syndrome

Prader-Willi syndrome illustrates genomic imprinting, when the gender of a parent is important.
I’m happy to see that fears about using CRISPR to edit human genes have dampened over the past year, but it’s still fun perusing the hyperbolic headlines:

They’re going to CRISPR people. What could possibly go wrong?

The Very Real Dangers of New Gene-Editing Technology

“How Gene Editing Could Ruin Human Evolution”

Since being fortunate enough a few years ago to find myself one of only two journalists in the press room at a genetic conference with two of CRISPR/Cas9’s inventors, I’ve made an effort to highlight exciting, beneficial uses of gene editing techniques, such as applications in "http://blogs.plos.org/dnascience/2017/06/29/can-crispr-conquer-huntingtons/">Huntington’s disease, sickle cell disease, and split-hand/foot malformation.

Another compelling study using CRISPR to interrogate a gene behind a disease appears in Human Molecular Genetics, from post-doctoral researcher Maeva Langouet, Marc Lalande, professor of Genetics and Genome Sciences and their colleagues at the University of Connecticut. The condition is rare, devastating, and has an unusual origin.

“Failure To Thrive” Leads to Obesity

The first signs Prader-Willi syndrome (PWS) aren’t especially distinctive or specific -- a small infant has poor muscle tone and is too weak to eat enough, leading to “failure to thrive.” By age 3, the child becomes better able to move around, and gains weight – and keeps gaining. The thinness of infancy and toddlerhood paradoxically becomes obesity, as metabolism slows. When the child becomes obsessed with seeking food, reflecting damage to the brain’s seat of satiety in the hypothalamus, the diagnostic odyssey may begin to focus in on Prader-Willi syndrome.

The overeating worsens, and there’s no pill or therapy or surgery that can cure the condition, although interventions address specific symptoms, and control of diet and exercise are crucial. But parents have to take measures that may seem extreme to those not in their shoes – they lock refrigerators, kitchen cabinets, and garbage pails to keep their children from consuming so much that their stomachs burst. Controlling the environment counters the inability to control the behavior.

PWS is the most common genetic cause of life-threatening obesity in children, but it has other symptoms. Young people with the condition may have poor growth, sleep apnea, intellectual and/or learning disabilities, sex hormone deficiency, and exhibit odd repetitive behaviors, such as skin picking.

Yet parents can channel some behaviors into positive actions. That’s the case for Jake Vasiloff, whose story appears in The Columbus Dispatch. Banging on pots and pans as a toddler, and then a toy bongo drum to get him to go to doctor appointments, led to today’s 18-year-old drummer. Music and art are terrific medicines for many children with rare diseases.

Altered Epigenetics

PWS arises from an intriguing short stretch of chromosome 15 that is subject to a phenomenon called genomic imprinting – the gender of the parent who contributes the glitch to the fertilized ovum (and therefore the child) is important.

Normally, methyl (CH3) groups cover the region of the chromosome that comes from the mother, effectively silencing it so that RNA isn’t transcribed and protein not translated. The individual is ok as long as the paternal part of chromosome 15 is okay, but if it’s missing, PWS results.

In 70% of individuals with PWS, about 5,000 DNA bases are deleted. Another 30% inherit both chromosome 15 sections from the mother (called uniparental disomy), and a small percentage of cases inherit PWS another way. About 1 in 15,000 newborns has PWS.

Interestingly, a nearby gene on chromosome 15 has the reverse imprinting pattern: the paternal copy is silenced and the maternal one works. If the maternal gene is missing, the child develops a different condition, Angelman syndrome. It causes autism and intellectual disability, an extended tongue, large jaw, poor muscle coordination, and characteristic arm flapping.

Lifting the Imprint

Theoretically, a way to counter PWS would be to turn on the silenced maternal gene. In late 2016, researchers from https://www.nichd.nih.gov/news/releases/122616-therapy-prader-willi">Duke University announced using that approach against a protein called G9a with promising results in mice.

Now the University of Connecticut team has silenced the maternal part of chromosome 15 a different way, in neurons from patients.

They worked with induced pluripotent stem (iPS) cells that were derived from skin cells of PWS patients. Adding a specific cocktail of biochemicals coaxed the stem cells to divide and give rise to daughter cells that specialized as neural progenitor cells and then brain neurons. The researchers zeroed in on a specific gene that encodes a protein called ZNF274, which normally tethers the silencing machinery to imprinted portion of the maternal side of chromosome 15. They deployed CRISPR/Cas9 to silence the ZNF274 gene so that there’d be no ZNF274 protein to shut off the maternal gene.

It worked. The treated nerve cells turned on the maternal copy of the Prader-Willi region of chromosome 15. So could ZNF274 protein be a drug target? Alas its functions are too “promiscuous,” binding to different sites, to reign in potential off-target effects. But researchers now know the next steps -- restrict the protein’s action to the part of the chromosome directly implicated in PWS.

For a disease without any treatment, a molecular correction is a promising beginning.

(Ironically, ZNF274 is zinc finger protein – enzymes that cut certain zinc finger parts of 1000+ proteins have been used experimentally to selectively silence genes since 2009, a forerunner to CRISPR so to speak. I wrote about zinc finger nucleases to treat hemophilia here.)

This post first appeared at my DNA Science blog at Public Library of Science.

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