Beware of Direct Lines

The most optimistic proponents of genomics suggest that with some human genomes (almost) completely sequenced, the next step of identifying disease-associated genes will be greatly enhanced. Now, so the argument goes, it should be possible to determine the functions of these genes and their corresponding gene products. Scientists hope this step will pave the way for the identification of drugs that target these disease-related gene products and treat or even cure the associated diseases. A scenario consistent with the above scheme appears to have been realized to an impressive extent with the new kinase inhibitor, STI-571 (Gleevec), which is being used to treat chronic myelogenous leukemia. However, other well-known examples of disease-associated genes and gene products raise doubts as to whether such a direct line from gene to therapy, let alone cure, may be typical or even common. Consider sickle cell disease. The pattern of inheritance (autosomal recessive) of sickle cell disease was first determined by James V. Neel in 1949,1 the same year that Linus Pauling and colleagues demonstrated that hemoglobin from sickle cell patients was physically distinguishable from hemoglobin from unaffected individuals.2 Eight years later, Vernon Ingram identified the amino acid substitution (valine for glutamic acid) at position 6 in the b chain that differentiated sickle hemoglobin from wild-type hemoglobin.3 Three years after that, Max Perutz and colleagues solved the three-dimensional structure of horse hemoglobin using X-ray diffraction.4 Now, 41 years later, the pathogenesis of sickle cell disease has been revealed to depend on a critical race between an astoundingly concentration-sensitive process of sickle hemoglobin polymerization, on the one hand, and erythrocyte transit and re-oxygenation on the other. If polymerization wins the race, what follows is a sorrowful cascade of further biochemical and physiologic events involving molecules, cells, tissues, and organs throughout the body. Diagnosis for sickle cell disease has certainly been facilitated by the availability of detailed nucleotide sequence information pertaining to the hemoglobin b chain genes, and by the remarkable elucidation of the pathogenesis. The effect on therapy is less clearcut. Current pharmacologic therapy does not target the sickle hemoglobin molecules directly. In fact, one of the few useful drugs, hydroxyurea, is believed to work by increasing the expression of fetal hemoglobin, neither polypeptide chain of which is encoded at the b-globin locus, the locus traditionally associated with sickle cell disease. The increased concentration of fetal hemoglobin is associated with a decreased tendency for sickle hemoglobin to polymerize, perhaps because the concentration of sickle hemoglobin is slightly reduced due to compensatory mechanisms. Sickle cell disease can be cured--and has been in a small number of severely afflicted patients--by bone marrow transplantation. The development of this approach has probably been facilitated by the availability of detailed information pertaining to the hemoglobin genes, but it does not depend absolutely on that knowledge. It is the risk and vast expense associated with this treatment, in conjunction with the rarity of sufficiently histocompatible donors for sickle cell sufferers, which limit the wholesale application of this treatment. What is most interesting is the nature of the relationship between gene product (hemoglobin) function and disease causation. Certainly, the most prominent biochemical function of hemoglobin is the binding, transport, and release of oxygen. While inadequate oxygenation of the tissues does play a significant role in sickle cell disease, the disease-associated mutation in the b chain does not directly have a major impact on the ability of individual molecules of hemoglobin to bind and release oxygen. The unfortunate consequence of the glutamic acid-to-valine substitution is that, when deoxygenated, the mutant hemoglobin molecules tend to self-associate into large fibers. These fibers in turn can distort the shapes of red blood cells sufficiently to compromise blood flow to tissues. It is the rate of this hemoglobin polymerization process that is key, because upon re-oxygenation, the growth of the fibers is abrogated and even reversed. There are no wonder drugs in routine clinical use for sickle cell disease that prevent or reverse this process of hemoglobin polymerization. The only drug on the horizon that actually decreases the probability of hemoglobin aggregation in vitro, nitric oxide, was discovered to have these properties empirically. If, in line with current molecular biological fashion, a massive screen of protein-protein interactions in human erythroid lineage cells had been undertaken, it is not clear that identification of noncovalent partners for hemoglobin would have improved therapy for sickle cell disease in the slightest. It is equally doubtful whether application of gene arrays to elucidate the expression patterns of the b-globin gene, likewise would add to the design of new "rational" therapies. Glib predictions of cures to flow from the successful genome sequencing effort are similarly brought into question by consideration of cystic fibrosis (CF). It was 12 years ago when the gene associated with CF was identified.5 As in the case of sickle cell disease, this information has led to clear improvements in diagnosis. However, the claims made years ago that the identification of the "CF gene" would lead inexorably, and quickly, to the cure look hopelessly naïve in retrospect. We lack not just a cure, but even a therapy that could be said to derive directly from the advance in genetic information. It is instructive to consider one further aspect of the quest for a curative approach for CF. The standard paradigm for rational drug development is that the cloning of a disease-associated gene leads to the structural characterization of the gene product. Then, structure-based design of a suitable ligand to activate or deactivate the gene product provides the cure. The allelic gene product most frequently associated with CF, the so-called delta508 allele at the cystic fibrosis transmembrane conductance regulator (CFTR) locus, is not actually expressed in significant quantities at the cell membrane, where it belongs.6 In fact, if speculations that the CF-associated allele in single dose provides protection from infectious causes of diarrhea are correct, its function is essentially the opposite of that of the product encoded by the wild-type allele: that is, not to transport chloride ions. Consequently, for many CF sufferers there is no gene product to influence by standard pharmacotherapy. A third informative example of the benefits and the limitations of gene characterization can be found in the field of clinical transplantation. Both solid organ and stem cell transplants are more successful when the recipient and donor express identical, or at least, similar alleles at loci known as histocompatibility loci. 7 The most important histocompatibility genes for human transplants are known as human leukocyte antigen (HLA) genes. Determination of the nucleotide sequences of several hundred alleles at these HLA loci was performed independently of the Human Genome Project over the past 15 to 20 years. This genetic information permitted the development and (now routine) use of DNA-based methods for the determination of the HLA alleles (HLA typing) expressed by transplant recipients and donors. DNA-based methods of HLA typing have been found to be more discriminating, accurate, and reproducible than the traditional antibody-based methods for HLA typing. The overall effectiveness of clinical transplantation programs has probably been substantially enhanced by these new gene-based methods of HLA typing. Nevertheless, the limitations of the knowledge of HLA genes are also apparent. Currently, knowing the nucleotide sequences of relevant genes can neither guarantee the availability of well-matched donors for many recipients, especially those with less common HLA alleles, nor guarantee sufficient numbers of donors at any level of recipient-donor HLA match.8 In fact, the disparity between the number of individuals awaiting transplantation and the number of donors available has increased every year that such data have been acquired. For many diseases or clinical conditions, by far the most important, and rate-limiting event leading to the development of effective therapy is the typically laborious and time-consuming unraveling of the relevant pathogenetic mechanisms, not the identifying, cloning, and sequencing of a relevant gene (or genes), even if the latter facilitates the former. How refreshing it would be if advocates of genomics could acknowledge this reality. Neil S. Greenspan, MD-PhD, is professor of pathology at the Institute of Pathology, Case Western Reserve University, Euclid Avenue, Cleveland, Ohio, 44106-4943 References 1. J.V. Neel, "The inheritance of sickle cell anemia," Science, 110:64-6, 1949. 2. L. Pauling et al., "Sickle cell anemia, a molecular disease," Science, 110:543-8, 1949. 3. V.M. Ingram, "Gene mutations in human haemoglobin--chemical difference between normal and sickle cell haemoglobin," Nature 180:326-8, 1957. 4. M.F. Perutz, et al., "Structure of Haemoblobin--3-dimensional Fourier synthesis at 5.5-A resolution, obtained by X-ray analysis," Nature 185:416-22, 1960. 5. J.M. Rommens et al., "Identification of cystic-fibrosis gene--chromosome walking and jumping," Science, 245:1059-65, 1989. 6. S.E. Gabriel et al., "Cystic-fibrosis heterozygote resistance to cholera-toxin in the cystic-fibrosis mouse model," Science, 266:107-9, 1994. 7. S.K. Takemoto et al., "Twelve years' experience with national sharing of HLA-matched cadaveric kidneys for transplantation," New England Journal of Medicine (NEJM), 343:1078-84, 2000. 8. B. Gridelli, G. Remuzzi, "Strategies for making more organs available for transplantation," NEJM, 343:404-10, 2000.

Beware of Direct Lines

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