Genetics: August 2008 Archives

Thanks to the Drug and Device Law Blog for providing a link to the opinion of New Jersey federal judge William Martini in the case of Gunvalson v. PTC Therapeutics. Comment on the reported opinion, which concerns access to an experimental drug for Duchenne muscular dystrophy (DMD), was made in a previous post, and follow-up information from a primary source is always welcome—particularly when plenty of questions about the case remain unanswered.

One notable issue raised in the opinion (page 7), which is pretty much dismissed by the judge, is whether the plaintiff, Jacob Gunvalson, even has DMD or, instead, a related and less severe condition, Becker muscular dystrophy (BMD). The distinction is important because the 2 conditions depend on the nature and consequence of the mutation in the gene that encodes the dystrophin protein. Deletion mutations in the gene that cause an out-of-frame mRNA transcript are likely to produce nonfunctional dystrophin, which manifests clinically (in general) as the more severe DMD. Patients with BMD are believed to produce partially functional dystrophin, which accounts for the milder illness.

The nature of Jacob Gunvalson's dystrophin mutation (if detectable), his dystrophin production, and the clinical severity of his illness are important (if not crucial) for 1) anticipating whether PTC's experimental suppressor of nonsense mutations, PTC124, is even likely to work; and 2) ensuring the clinical uniformity of enrollees in clinical trials (if patients with BMD are not eligible).

I can only assume that these issues were laid aside by the judge because either 1) he is incapable of understanding them or their importance; 2) they weren't argued sufficiently by the defendants; or 3) they're resolved issues in the minds of the defendants (meaning that Jacob Gunvalson is known to harbor a nonsense mutation and, indeed, had DMD). [See information in the Addendum below, which suggests that Jacob's diagnosis is not resolved.]

Alright, those fundamental medical concerns raised, back to the opinion:

The undisputed background on the case is that, in early 2006, PTC began a 28-day, open-label phase 2a trial of PTC124 in patients with DMD. At the time, Jacob was taking the well-known aminoglycoside antibiotic gentamicin, which is known to suppress nonsense mutations. (It is unclear how Jacob was receiving gentamicin: from his personal physician or through a clinical trial.) According to the opinion, Jacob's mother consulted PTC vice president Claudia Hirawat about whether Jacob should discontinue gentamicin, so that he could enroll in the PTC124 clinical trial. (Why Hirawat, who is evidently not a medical professional, would be consulted over Jacob's personal physician on this issue is not known, nor addressed, in the opinion.)

According to the affidavit of Jacob's mother, Hirawat told her that Jacob should continue the gentamicin, "which appeared to have some beneficial effect, and wait for later PTC124 clinical trials." This is the alleged exchange (along with similar exchanges) on which the entire case apparently rests: Whether PTC (as represented primarily by Hirawat) led Jacob and his mother to believe that Jacob would have access to the unapproved PTC124, even though he was advised not to and/or didn't enroll in the phase 2a trial of PTC124.

As it turns out, the phase 2a trial was a "success," as Judge Martini describes it. (Frankly it's hard to call such a small, early-phase trial a success—unless by success, one means "not a failure.") According to the PTC web site, enrollees with DMD and a nonsense mutation in the dystrophin gene received 1 of 3 dosages of PTC124 for 28 days. Data from 26 patients who received low or medium dosages were presented at the Third Annual Congress of Myology in May. 

Muscle biopsies showed evidence of dose-dependent increases in dystrophin expression and significant reductions in creatine kinase levels. Also parents and teachers reported clinical improvement in this non-blinded, non-placebo-controlled study, and the drug appeared to be reasonably well tolerated. Given the positive phase 2a trial results, a 2-year extension phase was initiated. 

At this point in time, Jacob's clinical status had deteriorated, according to the opinion, but it does not describe how. (News reports suggest that Jacob had become nonambulatory.) Consequently Jacob sought access to PTC124; however, he was not eligible to participate in the 2a extension trial, because he had not been enrolled in the 28-day study (which PTC had discouraged, claimed the Gunvalsons).* Therefore the Gunvalsons sought access to PTC124 through the FDA's compassionate-use program, which PTC refused to pursue. 

By my read, Judge Martini bases his opinion in favor of the Gunvalsons primarily on whether PTC created some kind of legal obligation to provide PTC124 to Jacob. The judge refers heavily, on this issue, to the affidavit of the patient's mother, Cherie Gunvalson, and what was said and/or promised to her by PTC employees, specifically Hirawat. However, Hirawat's statements, as quoted by the judge, provide an important caveat to clinical-trial enrollment that is overlooked (in my opinion) by the judge. For example,

I informed Mrs. Gunvalson that Jacob's non-enrollment in the phase 2a trials would not by itself preclude him from participating in all of PTC's anticipated future clinical trials, for PTC 124, assuming he satisfied the eligibility requirements for those trials [emphasis added].

It is also important (at least to me) that this statement does not promise enrollment in the 2a extension trial specifically (although some may consider this point too fine to care about). In addition, the accurate quotation of other statements allegedly made to Jacob's mother are suspect, in my mind. For instance, PTC chief medical officer Langdon Miller is claimed to have promised unqualified access to PTC124, a dubious proposition: "[O]nce positive results were back from the [phase 2a] trial, Jacob will get PTC124."

Lawyers Jim Beck and Mark Hermann, over at the Drug and Device Law Blog, dissect some of the legal issues in the case—such as the advisability of communications between company representatives and patients and the nontrivial nature of opening a compassionate-use program for an individual patient. They also make a lot of decent-sounding arguments for why Judge Martini's opinion is a raw deal for any company that develops drugs for hopeless conditions. 

* A previous post discusses Jacob's enrollment in a phase 2b trial of PTC124; however, a footnote in Judge Martini's opinion dispels any notions that either party thought Jacob was or is eligible for enrollment in this trial (see opinion footnote, page 3).

Addendum: In its press release on Judge Martini's opinion, PTC writes, "The decisions made about prior trials were decisions made by the Gunvalsons or the principal investigator for that trial, and not based on any promises or assurances by PTC. In fact, medical records and emails from the Gunvalsons also indicated that Jacob was ineligible for our Phase 2a trial because of the specific nature of his medical condition."

Evidently Richard Finkel, MD, principal investigator of the 2a trial of PTC124, believed that Jacob had BMD on the basis of a record review. A diagnosis of BMD would have disqualified Jacob from PTC's 2a trial, according to Finkel (see page 7 of Martini's opinion). However, another principal investigator, Brenda Wong, MBBS, MRCP, believed that Jacob had DMD. The judge conferred greater weight to Wong's diagnostic opinion.

In a more complete account of last week's "anthrax" science briefing by the FBI, USA Today describes how the incriminating flask of Bacillus anthracis at USAMRIID, RMR-1029, became so genetically distinctive.

RMR-1029 started out as spores from an original Ames strain isolate, which was obtained from a dead Texas calf in 1981. At the US Army's Dugway Proving Ground, 13 production runs were initially conducted with this Ames isolate. Then USAMRIID scientist Bruce Ivins ran another 22 runs, to produce 164 liters of spores in 1997. Later Ivins concentrated the spore collection, called at this time RMR-1029, to 2 flasks in 2001 and then one 1-liter flask in 2004.

Because RMR-1029 had therefore been produced from so many generations of B. anthracis (Paul Keim of Northern Arizona University estimated that one spore colony might represent up to a trillion generations, wrote the paper), subpopulations of spores in the collection harbored distinctive mutations.* Four of these mutations were used by investigators to trace the letter spores back to RMR-1029.

*Strains of B. anthracis are usually highly genetically conserved, because spores in the wild typically remain dormant in the soil for such a long period of time before growth in an infected animal.

Scanning electron micrograph of spores of Ames strain of B. anthracis from CDC/Janice Haney Carr.

Addendum: USA Today, unlike other media outlets, also provided the FBI's list of scientific publications that relate directly to the anthrax investigation.

• Read TD et al. Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science. 2002;296:2028-2033.
• Cummings CA, Relman DA. Genomics and microbiology: microbial forensics—"cross-examing pathogens." Science. 2002;296:1976-1979.
• Read TD et al. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature. 2003;423:81-86.
• Whiteaker JR et al. Quantitative determination of heme for forensic characterization of Bacillus spores using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem. 2004;76:2836-2841.
• Easterday WR et al. Specific detection of Bacillus anthracis using the TaqMan mismatch amplification mutation assay. Biotechniques. 2005;38:731-735.
• Beecher DJ. Forensic application of microbiological culture analysis to identify mail intentionally contaminated with Bacillus anthracis spores. Appl Environ Microbiol. 2006;72:5304-5310.
• Van Ert MN et al. Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain. J Clin Microbiol. 2007;45:47-53. (Discussed in a previous post, here.)
• Brewer LN et al. Forensic analysis of bioagents by X-ray and TOF-SIMS hyperspectral imaging. Forensic Sci Int. 2008;179:98-106.

Yesterday's FBI press briefing on the 2001 "anthrax" letter attacks was intended to rectify a few erroneous pieces of information in press reports and to bolster confidence in the science linking Bruce Ivins to the mailed Bacillus anthracis spores. However, several media outlets (eg, NYT) and Senator Tom Daschle, a target of one of the anthrax letters, continue to express skepticism that the FBI undeniably had its man. Specifically, to highlight the FBI's ineptitude, much is being made of an initial, unusable spore sample that was provided by Ivins to the FBI in 2002 and later destroyed by the agency.*

In an opening statement at the press briefing, Vahid Majidi, PhD, Assistant Director of the FBI's Weapons of Mass Destruction Directorate, clarified that the mailed spores had not been "weaponized" with silicon—contrary to numerous, previous reports. Specifically Joseph Michael, PhD, at the Sandia National Laboratories (who was present at the briefing), concluded that silicon had been naturally incorporated into the spores after examining them with transmission electron microscopy. Majidi also further outlined the genetic investigation that led to the RMR-1029 flask of B. anthracis at USAMRIID (as graphically presented in a recent post).

An initial, preliminary analysis of the letter spores at the CDC revealed a mixture of several phenotypes. This discovery led to the extraction of DNA from these phenotypic variants at Northern Arizona University and full sequencing of the DNA samples at The Institute for Genomic Research (TIGR). Majidi further advised that additional scientific information would be available in peer-reviewed publications and asked the audience to "respect the integrity of this process." He also acknowledged the FBI's inability to quell all suspicions related to the FBI's case against Ivins and added, "There's always going to be a spore on a grassy knoll," wrote the NYT.

Other panel scientists at the briefing included FBI Laboratory Director Chris Hassell, PhD; Paul Keim, PhD (Northern Arizona University); James Burans, PhD (National BioForensic Analysis Center); Rita Colwell, PhD (University of Maryland; Johns Hopkins Bloomberg School of Public Health), Claire Fraser-Liggett, PhD (University of Maryland); and Jacques Ravel, PhD (University of Maryland).

Meanwhile, Slate blogger Glenn Greenwald—evidently forsaking all other evidence—believes the FBI's case against Ivins is completely undermined by the agency's inability to pinpoint exactly when Ivins drove from USAMRIID in Fort Detrick, Maryland, to Princeton, NJ (160 miles), on 2 occasions to mail the anthrax letters postmarked 9/18 and 10/9 of 2001.

* However, Paul Keim's lab at Northern Arizona University reportedly kept a sample of these spores for later analysis.

Additional source: ScienceNOW Daily News.

Scanning electron micrograph of spores of Ames strain of B. anthracis from CDC/Janice Haney Carr.

Despite the discovery in 1993 of the altered gene that causes Huntington's disease (HD), treatment for this dismal illness remains purely symptomatic and supportive. In the United States, medical therapy for HD chorea has been limited to traditional dopamine-receptor-blocking neuroleptics; however, these medications are associated with a high incidence of extrapyramidal side effects (eg, Parkinsonism) and irreversible tardive dyskinesia.

On Friday, the FDA approved tetrabenazine (Xenazine; Prestwick Pharmaceuticals), a well-known dopamine-depleting agent, for the treatment of HD chorea. The drug, which was granted "orphan drug status," is the first medication approved in the United States for the movement disorder; although tetrabenazine has been in use for HD chorea in Canada, Europe, and other parts of the world. The agent, which probably has limited dopamine-receptor-blocking properties at therapeutic dosages, has not been associated with the development of tardive dyskinesia.

Tetrabenazine was approved for HD chorea on the basis of a multicenter, prospective, double-blind, placebo-controlled study of ambulatory patients with HD (N = 84). Randomly assigned tetrabenazine* was associated with a 3.5-unit relative reduction of the chorea score (Unified HD Rating Scale) at 12 weeks. Five tetrabenazine-treated patients withdrew from the study, and 5 serious adverse events—drowning suicide, complicated fall, restlessness/suicidal ideation, and breast cancer—were reported with treatment. Nevertheless, another controlled study (industry sponsored) and open-label studies support the drug's use in HD.

With the approval of tetrabenazine, the FDA requires a company-supplied Risk Evaluation and Mitigation Strategy (REMS) to manage any serious risks associated with the drug.

* Titrated up to a maximum of dosage of 100 mg/d.

Public-domain photo of American folk legend Woody Guthrie, who died of complications due to HD in 1967.

The techniques used to trace the Bacillus anthracis spores from the 2001 "anthrax" letter attacks to the USAMRIID laboratory at Fort Detrick, Maryland, weren't particularly novel, according to a report in this week's Science. By using documents released by the DoJ last week and expert speculation, writer Martin Enserink proposes the series of events that led to the source of the B. anthracis (Ames strain) that killed 5 people.

The first major task was to genetically distinguish the letter spores on the basis of phenotypic differences in cultured bacterial subpopulations, if at all possible.

The second task was to match the letter spore makeup to known, available Ames strain samples.

The FBI likely identified the USAMRIID lab as the source of the letter spores given that it was the only lab within the area where the "federal eagle" envelopes used in the attacks were distributed and sold. Although this very sound conclusion does not completely rule out the possibility that the spores came from another lab, it is the confluence of evidence (including Ivins's alleged submission of sabotaged or false B. anthracis samples to the FBI) that indicts the former USAMRIID scientist.

SNPs = single nucleotide polymorphisms.

* Sequencing work was likely performed by scientists at Northern Arizona University and The Institute for Genomic Research (TIGR) in Rockville, Maryland.

08/18/08 update: According to ABC News, 8 of the positive samples originated from 2 US labs. One is presumed to be USAMRIID, and the other is not named. The original (2002), "unusable" sample of B. anthracis provided by Bruce Ivins to the FBI—a portion of which was retained by Paul Keim at Northern Arizona University—later tested positive for the 4 "letter" mutations.

While we wait for confirmation and further explanation from the scientific community (and I suspect specifically from Northern Arizona University's Paul Keim) about the forensic evidence that ties Bruce Ivins to the 2001 anthrax attacks, we can comb the documents released today by the DoJ. At least from my perspective, the evidence against Ivins is very compelling.

Among the presented information is the fact that the Ames strain of Bacillus anthracis contained in the mailed letters can be directly linked to a single spore batch, called RMR-1029. This identification was accomplished by the detection of 4 characteristic genetic mutations, which are otherwise not described in the DoJ documents; however, it is possible that these mutations refer to the use of highly mutable single nucleotide repeat (SNR) markers described in a previous post here on the subject.

Of the 16 domestic labs that had RMR-1029 before the 2001 anthrax attacks, only one—the USAMRID facility in Fort Detrick, MD—was located where the identified "federal eagle" envelopes used in the attacks were distributed and sold (Maryland or Virginia).

At Fort Detrick, RMR-1029 was stored in the B3 biocontainment suite in Building 1425, to which Bruce Ivins had "unrestricted access." Moreover, Ivins had been "the sole custodian of RMR-1029 since it was first grown in 1997."

Because the B. anthracis spores in the letters sent to the Post and Tom Brokaw (postmarked 9/18/2001) were physically different* from those contained in the letters to Senators Leahy and Daschle (postmarked 10/9/2001), the investigators concluded that the spore batches were created from the RMR-1029 flask at Fort Detrick on two separate occasions. This conclusion is supported by the presence of a B. subtilis contaminant in the Post/Brokaw letters, which could not have been derived from the Fort Detrick flask.

On the basis of nighttime work records at Fort Detrick, the DoJ proposes that Ivins produced the Post/Brokaw B. anthracis during September 14-16 and the Leahy/Daschle spores sometime from September 28 to October 5. It is important to note that Ivins worked alone during these late-shift periods, and that his work time differed considerably from those of other Fort Detrick researchers with access to RMR-1029.

Probably most damning is the allegation that Ivins stalled the genetic identification of the RMR-1029 batch at Fort Detrick by submitting possibly sabotaged or false samples to the FBI for analysis. Initial samples provided by Ivins in 2002 were unusable, according to the DoJ documents, and a second sample of RMR-1029 from Fort Detrick did not contain the characteristic 4 mutations. In 2004, the FBI seized the RMR-1029 flask, which subsequently indicated a genetic match to the B. anthracis contained in the letters. 

A final note of curiosity is the return address provided on the Senators' letters: "4th Grade, Greendale School, Franklin Park, NJ, 08852." Former person of interest Stephen Hatfill had been loosely linked to the fictitious address, owing to erroneous media reports that he lived near a "Greendale Elementary School" while in Zimbabwe. The DoJ documents suggest that the return address may be an indirect reference to the Greendale Baptist Academy in Wisconsin and a related suit filed by the ultra-conservative American Family Association against the Wisconsin Department of Public Services, which was investigating the corporal punishment of a 4th-grade student at the Academy. The DoJ notes that "Mr. and Mrs. Bruce Ivins," who were practicing Catholics, made several donations over the years to the AFA and received the organization's newsletter, which would have referenced the suit.

* Genomic sequencing, however, revealed that the B. anthracis spores in all 4 letters were identical.

Scanning electron micrograph of spores of Ames strain of B. anthracis from CDC/Janice Haney Carr.