September 28, 2010 | In the fall of 1992, an M.D. from Johns Hopkins Medical School drove down to the National Press Building in Washington DC to interview for an editorial position with a new scientific journal called Nature Genetics. It was soon apparent to both parties that his interests and ambitions wouldn’t be met reading scientific manuscripts all day. The men shook hands and wished each other well.
I didn’t see Hugh Rienhoff again until 15 years later—on the cover of Nature, pictured with his young daughter. Beatrice Rienhoff suffers from an undiagnosed genetic syndrome that has stumped the best clinical geneticists in the country. But if his friends and colleagues couldn’t identify the cause of Beatrice’s condition, Rienhoff would do it himself.
“With a sequencer and a website, Rienhoff has stepped over the threshold of personal genomics in a way set to catch the imagination,” Brendan Maher wrote in Nature in 2007. “As sequencing gets ever easier and knowledge bases ever larger, it may not be fanciful to imagine more and more people following him, developing theories about abnormalities and testing them through sequencing.”
Five years of searching for clues in his daughter’s genome may have finally paid off. Rienhoff’s efforts have unearthed a candidate gene called copine-1, and while the evidence is still circumstantial, if confirmed it would be a prize more precious than anything he has sought in his eventful career.
RIENHOFF’s adventures in genetics began shortly after abandoning his plans to enter science publishing. He joined one of Silicon Valley’s top venture capital firms, New Enterprise Associates (NEA), and enjoyed a cameo role in Michael Lewis’ The New New Thing, shadowing billionaire Jim Clark, the founder of Netscape, Silicon Graphics, and Healtheon. He was also part of the Abingworth team that launched next-gen sequencing company Solexa. Finally it was time to branch out on his own.
“DNA Sciences got started in ’92 in my head,” says Rienhoff, as we renewed acquaintances after 15 years over breakfast near MIT. (Perhaps that’s why I sensed he wasn’t fully committed to becoming a science editor.) He became fascinated with DNA chips, more for their potential in doing genetic interrogations rather than measuring expression. In 1997, Rienhoff met Mark Chee, one of the founding scientists at Affymetrix, and suggested they start a company together. But by the time Rienhoff had lined up some financing and left Abingworth in April 1998, Chee was launching a company called Illumina.
Not all was lost that summer, however. On June 6, Rienhoff married Lisa Hane. At the wedding reception, Seth Harrison, a venture capitalist, took Rienhoff aside. “Whatever you’re doing, we’re good for $5 million,” he said. Rienhoff put his house on the market, packed up the car and headed to California.
Rienhoff pitched his company, originally called Kiva Genetics, as more of a hardware company than a discovery company. The hardware was a novel automated, microfabricated platform performing Sanger sequencing, with hundreds of capillaries each pumping out 500-600 bases in 15 minutes. He licensed some key sequencing technology developed by Jay Flatley and Molecular Dynamics, which had just been acquired by Amersham.
In January 2000, DNA Sciences held a Series B finance round at the height of the Internet bubble. Rienhoff smiles as he recalls “the bubble”—the term sheet specified the pre-money of $100 million and included a 1964 Sunburst Stratocaster—something he’d seen in a pawn shop in midtown Manhattan on the way to a VC presentation and had to have! He raised $50 million, but the VCs insisted Rienhoff remove the guitar clause.
The sequencing technology was merely a means to produce “an unfair, in-house advantage in discovery.” Rienhoff’s big idea was to recruit patients by going straight to the source. “Academics hoard their patients,” he says. “I decided I’d just go direct to the patient. That’s when we decided to launch DNA.com.” It took about a year to get all that together—build a secure room to store records, figure out how to get blood samples, build a web site and get IRB approval.
The pitch to consumers went like this: DNA Sciences will come to your house, collect your blood and a medical history, and perform genome-wide association studies for specific diseases. If you volunteer, you might be a control in one study, a test subject in another. And you will never be told the results. Rienhoff’s punch line was: “This is a preliminary study, not a validation study. If we find out you’re APOE4 or BRCA1, we’re still not going to tell you. Are you OK with that?”
DNA.com launched on 1 August 2000, and was greeted by a front-page story in the New York Times, which quoted Rienhoff as saying: “This is the first opportunity for six-pack Joe in the heartland who has diabetes to participate in a study.” Rienhoff admits it was a little flip in retrospect, but it turned out “six-pack Joe” wasn’t insulted at all (or maybe he doesn’t read the Times). The response was phenomenal. Phlebotomists visited volunteers in their homes or offices, drew blood, took medical histories, and sent in 10,000 samples in a matter of weeks. “In many ways, it’s what the Personal Genome Project is today—and it worked!” says Rienhoff.
But things started to go awry in the second half of 2000. The acquisition of Axys Pharmaceuticals and its 250,000 consented, phenotyped samples valued DNA Sciences at $400 million, but Rienhoff now had nearly 200 staff spread across five sites on both coasts and the U.K.
On January 3, 2001, Rienhoff filed the S-1 document, and waited… and waited. “The bankers said, ‘Don’t worry, we’ll get back to the New Economy,’” Rienhoff recalls. “It never happened.” Meanwhile, the company’s cash reserves were dwindling. By early summer, Rienhoff had given up going public, and he resigned in the fall. “I had an unbelievable time, it was really fantastic. I learned a shitload about things I thought I knew, but didn’t.”
‘MY DAUGHTER pulled me back into all of this,” says Rienhoff. From the moment he first held his baby girl in 2003, Rienhoff sensed something unusual about her. She had long feet, and her fingers were clawed. Having studied at Hopkins with McKusick, Rienhoff instantly worried whether his daughter might have inherited Marfan syndrome, the rare genetic disease that McKusick and others have suggested affected Abraham Lincoln.
Rienhoff’s quest to find a diagnosis for his daughter has garnered widespread media attention. In 2005, Rienhoff flew Beatrice, then 18 months old, to Baltimore to visit David Valle, a renowned medical geneticist at Johns Hopkins. Valle and colleagues examined Beatrice in a makeshift clinic, noticing that she had a forked ulvula (the flap of skin in the back of the throat). Ironically, Valle’s colleagues Hal Dietz and Bart Loeys had just published a report in Nature Genetics describing patients with a new genetic disorder, named Loeys–Dietz syndrome, characterized by a split uvula, but more disturbingly potentially fatal weakness of the aorta.
Rienhoff suspected he had seen several cases of Loeys-Dietz during his residency and mistaken them for Marfan’s. The average lifespan of Loeys-Dietz patients was just 27 years. But it turned out Beatrice did not carry a mutation in the relevant genes—the TGF-β receptors 1 or 2. In his attic office, Rienhoff read up on the huge TGF-β signaling pathway. A friend told him about myostatin, a closely related hormone to TGF- β, which regulates the size and number of muscle fibers. It was the next obvious candidate to try.
Taking matters into his own hands, Rienhoff spent $2,000 on a used PCR machine and other lab equipment, and began amplifying his daughter’s DNA. “If you can make a good soufflé, you can sequence DNA,” he joked, though he ended up outsourcing the sequencing part. There were some intriguing variants, but again nothing definitive. Still, given the circumstantial evidence incriminating TGF- β signaling, Beatrice’s cardiologist agreed to put her on a drug called losartan, a prophylactic measure to ward off cardiovascular complications.
To help others who might also be struggling to solve a mysterious genetic disorder in their family, Rienhoff created a Web site called MyDaughtersDNA.org, which allows people to share their own experiences and receive expert help. “There’s no place where people and geneticists share unsolved cases,” says Rienhoff. “It might be quixotic to think that physicians might take time to post cases… Physicians don’t really understand genetics and they’re too busy to learn.” 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.
That summer, Illumina agreed to sequence the transcriptome (copies of all the expressed genes) from the Rienhoff family of five—Beatrice, her brothers and her parents. After painstakingly wading through reams of sequence data in his attic for more than a year, Rienhoff finally 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.”
IN reviewing the transcriptome sequence data, Rienhoff paid 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 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: 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.) 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). And it had to be 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-β signal transduction or regulation, including, says Rienhoff, 32 other members of the TGF-β 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?
In late 2009, as Rienhoff trawled through Beatrice’s transcriptome, 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.” The variant is also associated with tenfold lower expression than the wild-type allele.
The variant satisfies all of Rienhoff’s criteria, but he 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.”
Not too much is known about copine-1, but it is highly conserved, suggesting some functional importance. One of Rienhoff’s collaborators is Alan Beggs, a geneticist at Boston’s Children’s Hospital. “He’s been my deep throat in muscle biology,” says Rienhoff. “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.”
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.”
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.” A hematologist by training, Rienhoff was drawn to the plight of patients with congenital anemias including beta-thalassemia and sickle-cell anemia, who eventually succumb to iron overload resulting from frequent blood transfusions. “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,” he says.
It’s just possible that the glitch Rienhoff has discovered in his own daughter’s DNA may result in a new standard of care as well. •
This article also appeared in the September-October 2010 issue of Bio-IT World Magazine. Subscriptions are free for qualifying individuals. Apply today.