For some parents, a physician’s advice to “just take him home and love him,” presumably letting nature take it’s most likely course, is just not acceptable. This blog has championed many such parents, who serve as catalysts for others.
New to rare disease territory is Orah Lasko, whose toddler Jacob not only has an exceedingly rare disease, but a highly unusual mutation behind it. With all of the media coverage of the high costs of new biotech-based treatments – gene therapy, targeted cancer drugs, monoclonal antibody-based drugs, antisense oligonucleotides – having such a double dose of rarity could be quite an obstacle.
But that’s not stopping Orah. Nor are the words of a neurologist who advised her to stop pursuing treatments.
The Diagnostic Odyssey
Orah Lasko’s pregnancy, her third, had been uneventful, with normal findings on the standard prenatal tests. Jacob seemed okay when he was born in September 2017, with minor feeding issues that went away. His small genitals didn’t set off any alarm bells.
But as the months went on, other things appeared. Or didn’t.
Low muscle tone made Jacob floppy, so that he couldn’t put any weight on his feet when Orah and her husband Avi tried to help him stand. Orah, who did graduate work in speech therapy and is a court stenographer, became alarmed when the boy wasn’t babbling. Jacob also had a prominent forehead and a big head, and by 13 months old, had yet to crawl.
And so began the diagnostic odyssey.
“When Jacob was around 9 months old, I went to the pediatrician and said to her ‘something’s not right,’” Orah recalls. “He had too many things wrong with him for this to be just delayed speech. It didn’t make sense.”
But with persistence, Orah was, finally, heard. “See a geneticist,” the pediatrician said.
The symptoms reminded the geneticist of Prader-Willi syndrome, but the test for that was negative. Ditto for Klinefelter’s syndrome. So too was a microarray test for chromosomal duplications and deletions.
“I don’t think we’ll find out what’s wrong, so go live your life and enjoy him,” Orah recalls being told.
“If this was your child, would you accept that as an answer? If you knew in your gut something was wrong?” she answered.
After doing some research, Orah convinced the geneticist to send a blood sample off for Jacob’s exome to be sequenced. The exome, the protein-encoding portion of the genome, holds the information behind 85% of single-gene diseases.
“Something was wrong and I wanted to know, now. I realized how much of an advocate you have to be for your child. A lot of doctors give up and if you don’t do your own research and go with your gut, you’re left with no answers,” Orah told me.
Exome Reveals Rare Mutation
Jacob had his exome test sent off when he was 15 months old, with samples from his parents too to see if whatever mutations he had were inherited or had originated in him. Getting results took 4 months, with Orah calling weekly for updates. Finally, the test results revealed an answer.
Although the geneticist, from a hospital near their home in Florida, told the distraught parents that the findings were worse than expected, he didn’t fully explain them. Fortunately, Orah found Olaf Bodamer at Boston Children’s Hospital, who translated the jargon in the lab report.
This image shows the coding region in a segment of eukaryotic DNA. Image credit: Smedlib via Wikimedia, CC-BY-SA-4.0
A key finding: Jacob’s mutation had happened in him – neither parent had it. It was dominant, meaning that only one copy of the gene was mutant. This may turn out to be crucial information for developing a treatment. And practically it meant that future pregnancies for the parents shouldn’t be at risk. But a new dominant mutation often introduces what geneticists call a “toxic gain of function” – something new, rather than something missing that could be replaced.
Jacob’s mutation is in a gene called USP7. Genes are in pieces, with exons that are transcribed into messenger RNA (mRNA) and then translated into the amino acid sequence of proteins. Introns are segments that are snipped out before the protein is made.
Cell’s “read” the genetic information 3 DNA bases at a time, each triplet specifying an amino acid. If a mutation disturbs a “splice site” where an intron is normally removed, a mangled protein, or none at all, results. Cells have machinery to recognize and destroy misfolded proteins.
USP7 mutated in the egg or sperm that went on with its partner to become Jacob. The gene consists of 35 exons. Something went awry at the boundary of intron 19 – a wrong DNA base appears in the sequence, an A (adenine) where a G (guanine) should be to create the signal for intron splicing. Jacob’s ultrarare splice-site mutation is part of one of the pair of his 16th largest chromosome.
A Block in Protein Recycling
Jacob has a condition that doesn’t yet have a catchy name: USP7-related-diseases (see Tess’s Tale: Social Media Catalyzes Rare Disease Diagnosis). A quarter of children never speak, and most have developmental delay, intellectual disability, and abnormal brain MRI findings. Other symptoms are the low muscle tone and small genitals that first alerted Jacob’s parents, and autism spectrum disorder, feeding difficulties, reflux, seizures, abnormal gait, low weight, and short stature.
At the microscopic level, USP7 protein is normally made in abundance in many tissues. The inability to synthesize it reverberates from a cellular back-up in misfolded protein processing to the whole-body constellation of symptoms.
Normally, USP7 assembles with three other types of proteins, and the group activates a protein called WASH, which turns on the cellular process for handling misfolded proteins. The aberrant folding happens when proteins don’t contort into their specific three-dimensional shapes as they peel off from the messenger RNAs that represent genes. Usually these renegade proteins don’t do what they normally would. USP7 is critical for overseeing that all-important folding.
The cells’ quality-control response to weed out misfolded proteins sends them from the membranous labyrinthine where they’re made (the endoplasmic reticulum) into the gel-like surrounding cytoplasm. Here they’re “tagged” with yet another protein, the wonderfully-named ubiquitin.
Misfolded proteins are stretched out and refolded, correctly, in proteasomes. Image credit: Jawahar Swaminathan and MSD staff at the European Bioinformatics Institute via Wikimedia, Public Domain
A misfolded protein tagged with just one ubiquitin may straighten itself out and refold correctly. But doomed proteins festooned with ubiquitins are escorted into bubbles, called endosomes, that in turn ferry them to a spool-like conglomeration of proteins called a proteasome. As the harpooned misshapen protein moves through the opening of a proteasome, it is stretched and then chopped up, like a log in a wood chipper. The original misfolded protein is dismantled, liberating amino acids that are recycled into new proteins.
While researchers often discover how a rare disease arises from affected families like Jacob’s and Tess’s, a report from 2015 zeroed in on USP7 disease from a different starting point. Yi-Heng Hao, of UT Southwestern Medical Center and colleagues, looked at databases of microdeletions, exome sequences, and symptoms, and found that variants of USP7 cause the same symptoms as mutations in a better-studied protein with which it associates (MAGEL2). Mutations that affect different proteins that interact in a shared biochemical pathway often cause the same spectrum of symptoms, like clotting disorders.
Action!
After the diagnosis Orah and Avi gave themselves a few days to cry, and then went into action.
Although Jacob’s condition is exceedingly rare, Orah quickly found Bo Bigelow online, the dad from the Tess’s Tale post linked to above. Orah organized community fundraisers for research; posted on Facebook, where I circuitously found her; spoke to a researcher at St. Jude’s working on a mouse model of USP7 conditions; and contacted pharmaceutical companies, all of which said USP7 wasn’t in the pipeline.
Orah thought first of gene therapy, but anything involving ubiquitin, as the very name suggests, would be too pervasive to want to try to disrupt. That is, a gene therapy that goes off-target could be devastating.
When I heard “splice site mutation,” I suggested antisense oligonucleotide (ASO) technology. That approach slaps a bit of DNA-like material onto the errant splice site, acting like a cloaking device and restoring how the gene is “read.” ASOs work for a few conditions, such as the recently FDA-approved Spinraza to treat spinal-muscular-atrophy. Antisense also makes sense for some patients with splice-site mutations causing familial dysautonomia.
But when Orah sent me Jacob’s exome results, the jargon was unfamiliar. The parameters for the site of the glitch in the USP7 gene includes a mysterious “+1G>A”. I knew a guanine had been replaced with an adenine, but I didn’t recognize the significance of the +1.
Fortunately, a researcher Orah’s dad contacted explained in an email that the +1 meant that the splice site had a G that had to remain a G for the intron to be removed. The resulting mRNA, with the A substitution, would be too large or too small. ASOs to patch the glitch wouldn’t work, nor do we yet have ways to correct splicing errors, he informed the distraught family.
Orah was at first devastated – she shares her thoughts frequently in beautifully written Facebook posts – but then chose to focus on the optimism in the kind researcher’s response. For his email went on to explain that the finding just meant they’d have to find another way. And then he offered specific suggestions.
One: study the copy of Jacob’s USP7 gene that is okay, figure out a way to express or overexpress it, and find out if doing so can override the mutation’s effect. That is, his genome has the instructions for the normal protein; find a way to access them.
Two: target or replace the USP7 enzyme with a protein therapy.
Three: tackle one of the proteins with which USP7 interacts.
Because genetics is an informational science, if one strategy to correct or compensate for misinformation derails, researchers can hypothesize others. But all of these clever approaches must first be tried in non-human animals, or in cultured cells or organoids.
The Bigger Picture
The story of a family’s quest for a way to counteract an extremely rare mutation behind an extremely rare disease illustrates the importance of basic research. Understanding the intron-exon make-up of genes is critical in the Lasko’s journey.
I remember when introns were discovered – something completely unexpected that was revealed with basic research. Walter Gilbert famously wrote an essay in Nature in 1978, “Why Genes in Pieces?” to explain the surprise. The essay is behind a paywall, but here’s a revisit from 2019. The discovery forever changed our view of the genes of complex organisms; most human genes are riddled with introns. Proteasomes, too, were discovered within the realm of basic research.
I hope that recent funding trends – more $ for the FDA, including the 21st Century Cures Act, but 12 percent less for the National Institutes of Health – doesn’t signal a trend in cutting off the very knowledge base and pipeline of drug candidates that fuel clinical trials.
For if gene therapy and ASOs won’t work to tackle Jacob’s disease, something else will, but not if we shift funding away from discovery. The loss of $80 million to the National Human Genome Research Institute for 2020, bringing funding down to $495 million, is horribly short-sighted.
Cures don’t come out of thin air. Cutting funding for basic research is never a good idea.
Source: PLOS EveryONE