Recently in Genetics Category

Fred Hassan, former CEO of the former Schering-Plough, believes that genomics will breathe new life into the pharmaceutical industry. His cheerleading prediction for how the study of the human genome will invigorate the flagging drug business, personalize healthcare, and extend lives is available at the HuffPo.

It's not an original thought, but Hassan (like the rest of us) recognizes that we're probably on the cusp of finally realizing tailored or boutique-style medicine. One case in point is the apparent, relative benefit and safety of the anti-amyloid bapineuzumab (JNJ; Wyeth) in individuals with Alzheimer disease who don't carry the ApoE4 allele.

In a more tangible way, genomics is informing the development of vaccines and the control of infectious diseases. In a recent PLoS review article ("The Key Role of Genomics in Modern Vaccine and Drug Design for Emerging Infectious Diseases"), Novartis employees describe how knowledge of pathogen and host genomes can be used to identify and select vaccine targets in a process known as reverse vaccinology.

As proof of this concept, the authors describe how whole-genome sequencing led to the identification of vaccine-candidate antigens against Neisseria meningitidis serogroup B. An 18-month analysis of the bacterial genome ultimately led to the identification of 570 genes that likely encoded surface-exposed or secreted proteins—more vaccine-candidate antigens than those discovered during 40 years of conventional analysis. Further antigen screening resulted in the development of a "multi-component" recombinant vaccine (Novartis's MenB vaccine), which is in phase 2/3 development.

A "potentially significant" mutation of the 2009 H1N1 virus has been detected in 3 severe cases (including 2 fatalities) of pandemic flu in Norway; however, the mutated virus is nothing new, according to the World Health Organization. The spontaneous mutation—which does not confer resistance to the antiviral drugs oseltamivir (Tamiflu; Roche) or zanamivir (Relenza; GSK)—was detected as early as April and has been found sporadically in both severe and mild flu cases in other countries,* reports WHO.

The 2009 H1N1 mutation has not been described or labeled explicitly in news reports. But according to a quoted Norwegian health official, the mutation "makes it possible for the virus to fasten itself or infect deeper into the bronchioles, and therefore provide for a more serious progress of the disease." This description suggests that the mutation lies in the gene encoding the viral binding protein, hemagglutinin.

WHO maintains that the current pandemic flu vaccine does produce immunity against the mutated virus.

* Namely Brazil, China, Japan, the Ukraine, and the United States.

Depiction of H1N1 virus from Wikipedia.

Although the gene responsible for Huntington's disease (HD), a dominantly inherited neurodegenerative disorder, was detected more than 10 years ago, the mechanism of its brain-specific pathology has remained elusive. Now investigators at Johns Hopkins reveal, in an elegant series of cell-line experiments, how the genetic disorder preferentially affects the corpus striatum, despite the fact that the mutant protein is found in cells throughout the body. Their findings are available in the journal Science. 

But first some necessary background...

HD is characterized by the expansion of a 3-nucleotide repeat section in the gene that encodes for the protein huntingtin (Htt). Neurotoxicity in HD is related to the cellular solubility of the mutant form of Htt, or mHtt. Protein aggregates of mHtt appear to be neuroprotective, whereas soluble mHtt is associated with cytotoxicity. It is also important to note that the mutant protein is sumoylated—meaning that it is covalently bound to a small ubiquitin-like modifier (SUMO)—which reduces neuroprotective protein aggregation.

Investigating Rhes protein...

The Hopkins investigators chose to examine the relationship between mHtt and the protein Rhes, because the latter is very selectively expressed in the corpus striatum. By using striatal (mouse) cell lines that overexpressed Rhes or that were Rhes deficient, investigators determined that Rhes binds "robustly" to endogenous Htt—but much more so to mHtt than to wild-type Htt. Moreover, overexpression of Rhes, in particular, profoundly affected the survival of cells that expressed mHtt (but not those expressing wild-type Htt). Specifically they found that the cytotoxic effect of overexpressed Rhes was concentration dependent. And in a final set of experiments, investigators determined that overexpressed Rhes augments the sumoylation of mHtt, thereby causing the disaggregation of mHtt and cell death.

So...it appears that striatal Rhes promotes localized neuronal degeneration in HD by enhancing the sumoylation of mHtt. Drugs that block the interaction between Rhes and mHtt may have therapeutic potential in HD, the authors conclude.

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

A newly discovered arenavirus, which caused fatal hemorrhagic fever in 1 Zambian and 3 South Africans* last year, has been named Lujo virus. The name is derived from the first 2 letters of the cities where victims manifested their illnesses: Lusaka, Zambia, and Johannesburg, South Africa. Lujo virus is the first Old World arenavirus discovered in 3 decades that is known to cause hemorrhagic fever, according to a new report in PLoS Pathogens.

A case of nonfatal hemorrhagic fever due to Lujo virus also occurred in a South African nurse, who cared for one of last year's deceased victims. The nurse evidently responded to ribavirin treatment and has completely recovered. Human-to-human transmission of Lujo virus is believed to be through contact with infected body fluids. How the index case acquired infection is unknown, but disease-causing arenaviruses are typically transmitted from rodent vectors.

* A Zambian safari employee (index case), a South African paramedic, a South African nurse, and a South African hospital employee.

Photo of house mouse (Mus musculus), which is the rodent vector for the Old World arenavirus, lymphocytic choriomeningitis virus, from Wikipedia.

In August, the FBI announced that USAMRIID scientist Bruce Ivins was the sole perpetrator of the "anthrax letter attacks" of 2001.

Pulmonary anthrax from inhaled letter spores killed 5 people, including 2 postal workers.

• Robert Stevens, 63, a photoeditor at a Florida tabloid office, died October 6, 2001.
• Thomas Morris, Jr., 55, a mail handler at the Brentwood mail facility in Washington, DC, died October 21, 2001.
• Joseph Curseen, 47, an employee at the Brentwood mail facility in Washington, DC, died October 22, 2001.
• Xinh Thi "Kathy" Nguyen, 61, a Vietnamese immigrant and New York City hospital worker, died October 31, 2001.*
• Ottilie Lundgren, a 94-year-old Connecticut resident, died November 21, 2001.*

Attention was directed to Ivins when DNA-sequence variations in the letter spores led to the identification of a specific flask at USAMIRIID, which contained the same mix of Bacillus anthracis spores. Ivins, a B. anthracis microbiologist, had been the primary custodian of the flask since 1997.

Other circumstantial evidence against Ivins:

• USAMRIID, in Fort Detrick, MD, is the only relevant lab in the geographic area where the "federal eagle" envelopes used in the attacks were distributed and sold.
• According to USAMRIID lab records, Ivins worked alone in late-night shifts during September 14-16, 2001, and from September 28 to October 5, 2001, before the rounds of Post/Brokow and Leahy/Daschle letters were respectively mailed.
• Ivins submitted "unusable" and false B. anthracis spores to the FBI for testing. The initial, unusable sample that was submitted to the FBI in 2002 was destroyed by the agency; however, Paul Keim's lab at Northern Arizona University preserved a portion of this initial sample, which was later found to contain mutations identical to the letter spores. In 2004, the FBI seized the USAMRIID flask, which subsequently indicated a genetic match to the B. anthracis in the letters.

Faced with impending prosecution by the DoJ, Ivins killed himself on August 1 July 29 by taking an overdose of Tylenol with codeine. (01/07/09 addendum: News reports now indicate that Ivins took a fatal overdose of acetaminophen, citing police records.)

Relevant background reading at the Pathophilia blog is available at these links:

• DoJ Evidence Against Ivins Compelling
• Genetic Gumshoeing in the Anthrax Letter Attacks
• Despite FBI Anthrax Briefing, Media and Blogging Arguably Egg on "Grassy Knollers"
• How the Smoking-Gun "Anthrax" Flask at USAMRIID Became a Smoking Gun

Also USA Today, believe it or don't, provided some of the most comprehensive news coverage of the bioterrorism investigation.

USAMRIID = US Army Medical Research Institute of Infectious Diseases.

* It remains unknown exactly how Nguyen and Lundgren were exposed to the B. anthracis letter spores.

Public domain photograph of Daschle "anthrax" letter from Wikipedia.

Three Generations, No Imbeciles: Eugenics, the Supreme Court, and

Buck v. Bell

By Paul A. Lombardo

365 pages

If generations of illegitimacy and limited intellect can be used to justify mass sterilization, then count on wiping out most of the southeastern United States, white or black or otherwise. But the consequence would not be merely the intended elimination of welfare-sucking trash—if such an outcome could be predicted. Say goodbye to the works of Tennessee Williams, Faulkner, Capote, and any other writer whose work draws on the freewheeling peccadilloes or horrors, depending on your viewpoint, of the extended Southern family. And say goodbye to anybody like yours truly, whose ancestral history in The Volunteer State contains its share of illiteracy, illegitimacy, and "imbecility" (thank you, US Census Bureau).

But unintended consequences were not considered by proponents of eugenics and sterilization laws in the early 20th century. In their conceit to claim an understanding of inheritance and Darwinian theory, eugenicists believed that the road to public health and lower taxes was paved with the legally mandated sterilization of society's feebleminded and promiscuous citizens—which, in their minds, meant men and women (but mostly women) of the lower classes.

At the center of this sorry time in American history is the legal case of Buck v. Bell, the end-result of Virginia's carefully crafted sterilization law, enacted in 1924. In comprehensive fashion, Georgia lawyer Paul Lombardo lays out this sad case, which was argued before the US Supreme Court, in Three Generations, No Imbeciles, the most detailed account to date of Buck v. Bell and its aftermath.

The Buck in this case was Carrie Buck, a young pregnant woman committed to the Virginia Colony for Epileptics and Feebleminded in 1924 under dubious circumstances. Carrie's mother, Emma, was already a member of the Colony under allegations of mental deficiency and moral turpitude, and Carrie's soon-to-be daughter would be placed in the foster care of the couple who arranged for Carrie's residence at the Colony. Among the 15 of so residents at the state institution selected as candidates by the Colony's Board for sterilization, on the basis of the newly enacted Virginia law, Carrie was chosen as the hapless test case. The argument for Carrie's sterilization would rest on flimsy evidence that 3 generations of the Buck family—including Carrie's infant daughter—constituted inherited imbecility.

Lombardo describes Carrie's legal defense, both at the state and federal levels, as a charade. Buck's anemic appeal before members of the Supreme Court, most of whom sympathized with the eugenics movement, led to a terse ruling in 1927, written by associate justice Oliver Wendell Holmes, Jr, which upheld the Virginia law. In his 3-page opinion, Holmes wrote the famous, cutting phrase, "Three generations of imbeciles are enough," which in fact had no foundation in the Buck family.

On the basis of the Supreme Court decision in Buck v. Bell, many other states (which already had sterilization laws in place) proceeded in relatively unfettered mode to conduct mass sterilization on their less fortunate citizens, and Lombardo writes that the US eugenics movement, bolstered by Buck v. Bell, informed a like-minded program in Socialist Germany, with known consequences.

Remarkably the Supreme Court decision of Buck v. Bell remains intact, although enduring sterilization laws in most states were repealed at the height of the civil-rights movement. (Laws in Washington state and Mississippi remain.) And while the general conceit of today's Americans may be that the constitutionality of legal sterilization won't be tested again before the high court, Lombardo reminds us that the same incentives to improve public health and lower tax burdens exist today and are manifest in selective reproduction methods and judge-mandated orders to not procreate.

For the first time, a predominant form of community-associated methicillin-resistant Staphylococcus aureus (MRSA) in the United States, USA300, has emerged as a major clone of MRSA in another country. In the latest issue of the NEJM, investigators from Colombia and Texas report their identification of USA300 isolates in a series of Colombian MRSA outbreaks, beginning in 2005. The researchers also discovered, in more than 2 dozen Colombian cases, a clone of vancomycin-resistant Enterococcus faecalis (VREF) that had been seen only in Houston.

The NEJM correspondence suggests that, although USA300 has been discovered as a predominant form of MRSA only in Colombia, the clone (not yet genetically identified) may well be a major cause of outbreaks in other Latin American countries.

Photo of spontaneously draining, cutaneous MRSA abscess on hip of prison inmate from the CDC. (Clarification: The photo, not the prison inmate, is from the CDC...at least as far as I know.)

A respite from the sad chain of questionable studies on autism is provided by this week's NEJM.

Investigators from the UK and UCLA report their discovery of 9 DNA variations (ie, single-nucleotide polymorphisms, or SNPs) in specific regions of the CNTNAP2 gene,* which are associated with a particular language impairment in nonautistic children of families affected by language disorders. These SNPs were found to associate in a highly significant way with the heritable behavioral marker (or endophenotype) of nonsense-word repetition.

But what's really interesting is that these SNP regions in CNTNAP2 have also been implicated (by some of the same investigators) in language delays among children with autism (Alarcon et al. Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Am J Hum Genet. 2008;82:150-159).

The NEJM authors conclude that similar DNA variations in these regions of the CNTNAP2 gene may predispose individuals to isolated language impairment, or they may contribute to the language impairment in autistic spectrum disorder. In the case of autism, other behavioral features, like impaired social interactions or repetitive behaviors (ie, stereotypy) are proposed to be caused by different genetic mechanisms.

These findings support the prevailing idea that autism, and the observed spectrum of the disorder, is the clinical manifestation of more than one complex, genetic alteration (see Happe F et al. Time to give up on a single explanation for autism. Nat Neurosci. 2006;9:1218-1220).

* CNTNAP2 encodes CASPR2, a neurexin, which is expressed in the developing human cortex. The authors identified CNTNAP2 as a target of the transcription factor FOXP2. (FOXP2 downregulates CNTNAP2 expression.) Mutations in FOXP2 are associated with speech and language disorders.

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.