By Kevin Davies
August 24, 2010 | EXCLUSIVE
Hugh Rienhoff’s relentless and inspiring search for the genetic basis for his daughter’s mysterious congenital disease has unearthed a strong candidate for the disorder.
With a sequencing assist from Illumina, help from a “volunteer guerilla” network of scientific friends, and the resolve to painstakingly wade through reams of sequence data in his attic for more than a year, Rienhoff has found suggestive evidence implicating a mutation in the gene for copine-1 (CPNE1) as the cause of Beatrice’s condition. But he’s the first to admit there’s still much more work to be done.
“It’s important for me not to represent that this gene is causative,” stresses Rienhoff. Nevertheless, he adds: “I’d rather have the whole world working on this gene right now.”
Rienhoff, a physician and biotech entrepreneur, has enjoyed a rich and eventful professional career. As a venture capitalist in the 1990s, he played a cameo role in Michael Lewis’ best-seller, The New, New Thing, shepherding Netscape co-founder Jim Clark. In 2000, he was the CEO of DNA Sciences and the mastermind of a website called DNA.com, a short-lived consumer genomics/sequencing outfit.
It was in 2007 that Rienhoff’s personal genomic quest first made headlines when he and his daughter graced the cover of Nature. Reporter Brendan Maher told the story of little Beatrice Rienhoff, born with a new and unrecognized congenital syndrome somewhat reminiscent of Marfan syndrome, and her father’s unwavering commitment to find the genetic cause. Rienhoff created a web community called MyDaughtersDNA.org to help other families share stories of undiagnosed patients and enable physicians and clinical geneticists to offer suggestions.
Five years ago, Rienhoff took his daughter to Johns Hopkins Medical School, where he had trained, to meet a team of medical geneticists led by David Valle, Bart Loeys and Hal Dietz. They tentatively concluded that Beatrice had a new syndrome named eponymously after Loeys and Dietz. The bad news for Rienhoff was that a hallmark of this syndrome was aortic malformation and premature death.
Beatrice’s major clinical features are very low muscle mass and certain facial features suggesting a midline developmental abnormality. Both are connected to flaws in TGF (transforming growth factor)-beta signaling. Beatrice’s condition did not fully match that of Loeys-Dietz syndrome, however (nor did she have mutations in the known genes), but Rienhoff was convinced that his daughter had to have a related disorder involving – like Marfan and Loeys-Dietz syndromes -- a defect in the TGF-beta pathway, or a related pathway involving muscle.
Usual Suspects
Rienhoff was – and still is -- determined to follow the project from its conception (literally) to the logical conclusion of pharmacologic management, assuming that is an option. “You start with a floppy little girl at birth and you have to remake yourself as a cell biologist,” he says.
With a little help from his research friends and a second-hand PCR machine, Rienhoff began studying candidate genes in the TGF-beta pathway. As described in a January 2009 article in Wired magazine by Brendan Koerner, the first few candidate genes (the myostatin receptors) proved uneventful, but that didn’t diminish his interest in the pathway.
Meanwhile, in the summer of 2008, Illumina agreed to sequence the transcriptome (copies of all the expressed genes) from the Rienhoff family of five -- Beatrice, her brothers and her parents. (Rienhoff has ties not only to Solexa, which Illumina acquired in 2007 – he briefly helped the venture team that originally set up Solexa – but has known Illumina CEO Jay Flatley since the late 1990s, when he licensed some sequencing technology from Flatley’s then company, Molecular Dynamics, for his own start up, DNA Sciences.)
Rienhoff set about analyzing the sequence data, paying particular attention to the new variants in Beatrice’s genome and instances where she was homozygous (carrying two copies) of the same variant, or allele. There were two logical possibilities: either Beatrice’s condition resulted from a new (de novo) mutation in her genome, or that she had a rare recessive disorder, inheriting single mutations in the same gene from her parents.
Rienhoff tentatively ruled out the former possibility. “We did find one bona fide new mutation but it was a very conservative amino-acid change in a protein that has relatively unknown function in humans and biologically wasn’t plausibly related to her clinical condition.”
That left Rienhoff searching for a candidate gene that had to satisfy three criteria:
1) It had to be a homozygous allele – Beatrice had to have two mutated copies of the same gene. (Technically, she could also be a compound heterozygote, carrying different mutations in both copies of the same gene that would have the same effect.)
2) The gene’s expression had to be altered in some way (for example by splicing, nonsense-mediated decay, or a mutation in a promoter or enhancer region.
3) It was on his candidate gene “short list” by virtue of some sort of connection with TGF-beta.
The problem was that Rienhoff’s short list was pretty long. There are hundreds of genes that encode proteins involved in TGF-beta signal transduction or regulation, including, says Rienhoff, 32 other members of the TGF-beta ligand family and five known receptors. Moreover, Rienhoff found his daughter had 932 instances of homozygosity. Which one, if any, could account for Beatrice’s disorder?
A Fair Cop?
In late 2009, as Rienhoff trawled through Beatrice’s transcriptome data, one of those homozygous gene variants stood out. “It took God-damn forever to find it. I had to sift through everything by hand,” says Rienhoff. “It was basically just brute force, hand-to-hand combat with this data.”
The allele in question is the insertion of a single T nucleotide in the gene for copine-1 on chromosome 20. Copine-1 is a protein involved in one arm of the TGF-beta signaling cascade. The insertion disrupts the gene’s reading frame and results in premature termination. “So that was cool,” says Rienhoff. “It was the only candidate with an insertion allele causing frameshift and nonsense-mediated decay signals.”
Not surprisingly, the variant is also associated with tenfold lower expression than the wild-type allele. “So that was nice,” says Rienhoff, adding that a tenfold reduction is significant by any measure.
The variant satisfies all of Rienhoff’s criteria, but quickly dismisses any suggestion that his search is over.
“Obviously I can’t say that [this gene] is it. That would be ridiculous, and I’d be stoned by the genetics community if I made such a statement. Nevertheless, I think it is a strong candidate.”
Rienhoff says copine-1 has not been well studied in humans or any other organism for that matter. It is highly conserved, however, indicative of some functional importance. One of Rienhoff’s collaborators is Alan Beggs, a geneticist at Boston’s Children’s Hospital (Harvard Medical School). “He’s been my deep throat in muscle biology,” says Rienhoff.
“By studying the rare, and possibly unique condition in his own daughter, Hugh may have discovered something of basic importance that may also have implications for a much broader group of patients,” Beggs says. “Of course, the ability for a single individual to direct this kind of research into their own genetics is truly transformative for how we view genetic studies.”
Aside from ongoing population genetic studies on the gene, Beggs has a panel of undiagnosed patients with clinical muscular problems, including some with conditions that resemble Beatrice’s, which he intends to resequence for possible copine-1 mutations.
Illumina is currently sequencing the exome (the protein-coding regions in the genome) for the Rienhoff family. “We’re probably the most studied family genomically on the planet,” says Rienhoff. “We’ll have the transcriptome, the exome, low-pass sequencing and we’ve done the “super chip,” the 1.4-million SNP chip.”
Rienhoff has convinced another friend to knock out the copine-1 gene in mouse muscle precursor cells. “I call him every Friday and ask him, ‘How’s it going?!’ It’s all stolen time, as you know.”
Iron Man
Since creating the MyDaughtersDNA.org Web site, Rienhoff says its use has been lighter than he might have anticipated, but that’s in part because many interested parties query him directly, either because they’re shy or have poor English or don’t want to publicly talk about their families. The most gratifying result came in 2008, when a Bulgarian family posted information about their 12-year-old undiagnosed daughter who had no tears. An American geneticist quickly suggested a diagnosis: Triple-A or Allgrove syndrome.
With his small network of friends – researchers and students at Washington University, MIT, Singapore and elsewhere -- Rienhoff has carried out transcriptome analysis on six other patients, usually children with a suspected sporadic or recessive genetic disease. He calls it “volunteer guerilla genomics.”
Rienhoff’s biggest concern over his daughter’s health is cardiovascular disease. A few years ago, Beatrice started to take the drug losartan, which she is tolerating well. The concern is that by down-regulating TGF-beta signaling to prevent vascular disease, the drug might be influencing Beatrice’s postnatal muscle mass. “It’s one of these Faustian deals, you know? I have to pick the worst evil to mitigate,” says Rienhoff. “She has gotten stronger but her overall weight is still low.” Rather than tinker with the drug dosage now, Rienhoff’s priority is to solve the molecular mystery underlying his daughter’s condition, so that he and Beatrice’s doctors can rationally decide the best therapy.
When he isn’t poring over sequence data in his attic, Rienhoff is planning clinical trials for his latest venture, Ferrokin Biosciences -- or as he calls it, “my day job.”
Rienhoff originally trained as a hematologist, and knows better than most that patients with congenital anemias including beta-thalassemia and sickle-cell anemia, eventually suffer from complications of the regular blood transfusions therapy. “They all die of iron overload. The name of the game is getting rid of the iron,” says Rienhoff. “These are people who could live normal lives.” But existing iron chelators either involve lengthy hospital infusions or toxicity and other side effects.
Rienhoff’s venture-backed company has licensed a family of novel compounds from academia and is already in phase II clinical trials for beta-thalassemia patients. “This could be standard of care,” says Rienhoff.