The first e-mail arrived on a Thursday morning, October 16, 2014. It was from a father in the Netherlands, sent to an eclectic list of researchers. Marc Pieterse had included me because he reads DNA Science
, my weekly blog at Public Library of Science.
“Dear Dr. Lewis,
My son Vincent (16-12-2002) has numerous small syndromic (dysmorphic) features and developmental delay. See the list below …”
What followed were seemingly unrelated oddities that would alert any clinical geneticist:
low nasal bridge, arched eyebrows, bluish eye whites, oddly-shaped low-placed ear
extra teeth, webbed neck, small lower jaw, elastic skin, brittle hair, loose joints, single crease in the palms
autism spectrum disorder, developmental delay, learning disability.
“To find out what the cause is, we started, years ago, with chromosomal and gene testing at the University Medical Center Nijmegen, genetics department. In the last test (whole exome sequencing) they did, his DNA was compared with the DNA of me and my wife, a so-called
“de novo” mutation on
RPS23 was found.”
Marc and his wife Anita do not have the mutation, which is in the gene that encodes one of the proteins that make up ribosomes, the snowman-shaped structures on which messenger RNA transcripts are translated into the amino acid sequences of proteins. A ribosome has 80 types of proteins distributed into a small and large part. “RPS” means ribosomal protein, small subunit, the top of the snowman.
Marc followed the learning curve of parents-turned-scientists, and quickly found Diamond Blackfan anemia (DBA)
that arises from a mutation in a different ribosomal protein, RPS19. Not exactly the same thing.
“No disease or syndrome is known to be directly related with RPS23,”
Marc concluded in that first of many e-mails to the group. He ended with a request:
“What is your opinion of this? Do you also do research on ribosomal gene mutations? Is there an alternative way to find out whether this mutation is the cause of my son’s condition?”
The conversation began immediately, as the dozen investigators, from all over the world, offered to help. Alyson MacInnes, PhD, now at Academic Medical Center in Amsterdam, and Susan Baserga, MD, PhD, who investigates ribosome biogenesis at Yale
, played crucial early roles in establishing the pathogenicity of Vincent’s mutation. Meanwhile, Marc focused on organizing the model organism communities -- yeast, bacteria, flies, frogs, mice, and zebrafish.
VINCENT’S EARLY YEARS
Marc had sensed something wasn’t quite right even before Vincent’s birth. In the bathtub, Anita’s belly would ripple wildly with the choreography of an energetic fetus. That activity didn’t let up as birth neared, and Marc ended up delivering his son in their bedroom.
The baby seemed well at first, although he was often sleepy, and spit up frequently. Then he began to lag in hitting developmental milestones.
At school Vincent acquired diagnoses of mild autism and nonverbal learning disabilities. Finally, at age 8, a pediatrician glimpsed the possible puzzle pieces. “She told me that Vincent has beautiful eyelashes, a cute face and nose, but little things that were different and not ok: hearing on one side was less, one ear was different and lower, one hip was higher. She advised us to check it out with a clinical geneticist,” Marc recalls.
Like Dorothy stepping onto the yellow brick road, Vincent began the diagnostic odyssey. Chromosomes were okay, nothing extra or missing, and the X wasn’t fragile. Nor did Vincent have Noonan, Kabuki, or Wiedemann Steiner syndromes, tests given in a round-up-the-usual-suspects approach to ruling out neurological conditions. Nothing relevant appeared on any branches of the family tree, even among Vincent’s brother and two sisters.
Then Marc read about exome sequencing – the 1% of the genome that encodes protein – and that led to Dr. Joris Veltman, PhD, head of the division of genome research at the Institute for Molecular Life Sciences in Nijmegen, who was already looking at kids with undiagnosed diseases
. After much cajoling, for Vincent wasn’t really ill, his exome was done. Then after waiting a year, the results were disappointing: “We didn’t find anything … but there was one mutation, but we don’t know much about that, it is unrelated
” to the symptoms.
Only it wasn’t.
It took 10 minutes for Marc to search using his son’s symptoms and the mutation and find Diamond-Blackfan anemia
. But that seemed to be a blind alley – Vincent doesn’t have anemia. Did he have a novel “ribosomopathy”? A “polysome profiling” test indeed showed that Vincent doesn’t make enough of the small subunit of ribosomes, which should hamper the ability of his cells to synthesize proteins.
A SECOND CASE
Years passed as Marc connected himself to anyone in the world working on ribosomopathies.
In September 2015, he registered Vincent’s mutation at GeneMatcher
, a website to connect researchers and clinicians with shared interest in a specific gene – but family members can enter data too.
“Today I received confirmation that a second heterozygous mutation was found in a patient in the Netherlands with strikingly 'overlapping' syndromic features and autism,” Marc e-mailed me on December 4. The second child echoed Vincent’s peculiarities, from the facial features, including the gorgeous eyelashes, to the single palmar crease, to the offset ears and autism. One striking difference: a split uvula, something seen in 2% of the general population and a sign of the inherited Loey-Dietz syndrome.
Unlike Marc and Anita, the parents of the second child do not want their child’s mutation to be researched, and the clinician won’t share it without their permission. So Marc has entered the world of bioethics, and the sharing of genomic information for the greater good.
“I think the rules on this are not correct. Any newfound mutation should be shared anonymously in the clinic and in research. A single mutation cannot be seen as a patient’s property. A total genome or DNA sequence is different; it is your identity. Because of the lack of a good set of rules, a lot of doctors stay on the safe side and don't disclose,” he wrote.
Not sharing DNA information hinders development of a simple single-gene test to find others – and the population of individuals with autism without a known cause is huge. “I am sure we will find more RPS23
mutations in the near future. Even knowing that the causative mutation is present, even without knowing the exact disease etiology, gives relief,” Marc emailed.
FROM HAIRS TO MODELS
Marc pursued all aspects of Vincent’s intriguing phenotype, including his thin, brittle hair. In January 2015, he found the recently-published article “The structure of people’s hair
” and contacted author Maikel Rheinstädter, PhD, who studies membrane biophysics at McMaster University and uses several techniques to interrogate hairs.
Dr. Rheinstädter responded right away, and soon Marc sent him hairs plucked from the family. “It's a long shot but it can be informative, and it is painless ;-),” Marc e-mailed me. Atypical hair is a feature of a few inherited disorders, including monilethrix
, Menkes disease
, and giant axonal neuropathy
A month later, Dr. Rheinstädter sent Marc a preliminary analysis and by September had finished a manuscript, with Marc as a co-author: “Structural Abnormalities in the Hair of a Patient with a Novel Ribosomalpathy: A Biophysical Case Report,” with lead author PhD student Richard Alsop. The tiny keratin spiral staircases of Vincent’s hairs’ appear normal, but they are about a fifth thinner than they should be, with a fifth fewer lipid molecules in the cell membranes, the continuous layer that cements the hair. I wonder if lipid deficits in neuroglia might explain the autism by similarly depleting the protective myelin sheath of axons.
Model organisms are becoming available too for Vincent’s syndrome. Dr. Baserga uses yeast to study ribosome biogenesis
. In Julia Dallman, Ph.D’s lab at the University of Miami, graduate student Sureni Sumathipala is using CRISPR/Cas-9 to silence the zebrafish version of Vincent’s gene and to insert his mutant variant. The highly social organism is used to study autism and to study Diamond-Blackfan anemia
. Matthew Cockman, PhD, a biochemist at the University of Oxford, and Jean-Paul Vincent, PhD
at the Francis Crick Institute, and others are developing fruit fly models. An RPS23
mouse will come from Nahum Sonenberg, PhD
, at McGill University. And so far five PhD theses will come from Vincent’s ribosomopathy, which doesn’t yet have a distinctive name.
FROM BIOCHEMISTRY TO THE BIGGER PICTURE
Model organisms with RPS23
mutations may untangle the web of oddities and symptoms that emerge from impaired ribosomes, but basic biochemistry will reveal the source of the pathology. When Alyson MacInnes, the early facilitator of the amazing network that Marc’s initial e-mail catalyzed, talked to Dr. Cockman, the pieces began to fit. He uses high resolution mass spectrometry on Vincent’s immortalized B cells. Dr. MacInnes explains:
“Dr. Cockman investigates an enzyme that places an oxygen atom onto the RPS23 protein, at a site just five amino acids away from where Vincent’s RPS23 has a lysine instead of an arginine, thanks to his single-base mutation. His RPS23 protein can mature and fold normally, which is why some of the researchers didn’t think he actually has Diamond Blackfan anemia. But the abnormal protein’s subtle structural difference interferes with how the ribosome reads “stop” signals in the messenger RNAs it translates – resulting in proteins that are too long. That is, Vincent’s glitch doesn’t just affect RPS23, but it affects translation of other
proteins too. And that raises a much more general and profound question: Do untrimmed proteins, caused by mutations affecting ribosomal proteins, cause autism and developmental delay in others?”
A fundamental defect in protein synthesis might explain why it hasn’t yet been possible to develop induced pluripotent stem cells from Vincent’s cells. Perhaps the small-subunit-deficient ribosomes can’t handle the surge in transcription required to hurtle skin fibroblasts back to an earlier developmental state.
While all of this research is going on, Vincent is active, happy, and loves nature, especially predator birds, and can identify and name more species than most adults, says Marc. He does judo, loves doing flips on a trampoline, and rides a mountain bike to school through a nature reserve, watching for birds along the way. He draws birds and other animals, and likes to play Minecraft
, Marc says (Vincent approved this description).
I asked Marc why he pursued the cause of his son’s collection of traits so relentlessly. “By finding out more about the mutation, I hope to learn more about the disease. Also when Vincent wants to have children, I want him to know the risks are 50% for passing on the mutation, and what kind of advice I should give him.”
But Marc Pieterse’s determination to drive research to identify his son’s spectrum of anomalies may go far beyond better understanding Vincent. It may in fact open a window to a much more general cause of autism – impaired protein synthesis. And considering the prevalence of autism, RPS23 disease may turn out not to be rare at all – even though now, only two patients are known.
On this Rare Disease Day 2016, I’d like to honor Marc, in the tradition of Augusto and Michaela Odone and their pursuit of understanding their son Lorenzo’s rare genetic disease more than two decades ago. Marc is one of the many parent heroes in the rare disease community. Kudos to you all!