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because it seems to be genetically indis- comparing both transcripts in the to a target for therapy. Thus, the genetic tinguishable from other variants embed- same sample, both methods decrease the community continues to require more tools ded in this haplotype block. The advent of effects of environmental and other con- to efficiently pinpoint risk variants that whole-genome association studies using founding factors. They also have advan- affect . There is no doubt the haplotype map11 will probably impli- tages over in vitro techniques, such as that many more inventive approaches will cate a substantial number of new disease transient transfection assays with allele- be developed for this. Ultimately, the associations with indirect markers that specific promoter constructs, as these stud- manipulation of regulatory mutations that are in linkage disequilibrium with rSNPs. ies are executed outside of their normal affect expression levels should be easier environment. than repairing or modulating the effects of Associations in search of a cause The haploChIP assay is useful as a surro- an abnormal . „

Providing that Pol II (or another DNA- gate marker of allelic variation in gene tran- 1. Cargill, M. et al. Nat. Genet. 22, 231–238 (1999). binding protein with similar properties) is scription, although it does not directly 2. Sachidanandam, R et al. Nature 409, 928–933 (2001). equally useful for testing any gene, identify the cis-acting polymorphism or 3. Brem, R.B., Yvert, G., Clinton, R. & Krugylak, L. haploChIP has the potential to allow the mechanism that is responsible for this vari- Science 296, 752–755 (2002). testing of each gene contained in a risk ation. This is not surprising, as there are 4. Cheung, V. et al. Nat. Genet. 33, 422–425 (2003). 5. Wasserman, W.W., Palumbo, M., Thompson, W., haplotype block for differential transcrip- many SNPs on the haplotype associated Fickett, J.W. & Lawrence, C.E. Nat. Genet. 26, tional activity. HaploChIP requires that with higher LTA transcriptional activity. 225–228 (2000). 6. Rockman, M.V. & Wray, G. Mol. Biol. Evol. 19, suitable cells or tissues that express the gene The task is also difficult because of the 1991–2004 (2002). are available and that the regulatory variant extensive distances at which transcriptional 7. Knight, J.C., Keating, B.J., Rockett, K.A. & 14 Kwiatkowski, D.P. Nat. Genet. 33, 469–475 (2003) affecting the test gene is heterozygous. control elements can be found . Should 8. Ozaki, K. et al. Nat. Genet. 32, 650–654 (2002). Another surrogate approach to in vivo human geneticists care about finding the 9. Daly, M.J., Rioux, J.D., Schaffner, S.F., Hudson, T.J. & Lander, E.S. Nat. Genet. 29, 229–232 (2001). transcriptional activity is allele-specific true regulatory SNP when the risk gene is 10. Rioux, J.D. et al. Nat. Genet. 29, 223–228 (2001). RT–PCR12,13. This is probably easier to identified and there already are many highly 11. Gabriel, S.B. et al. Science 296, 2225–2229 (2002) 12. Yan, H., Yuan, W., Velculescu, V.E., Vogelstein, B. & carry out but has an extra requirement that correlated SNPs that tag the disease haplo- Kinzler, K.W. Science 297, 1143 (2002). http://www.nature.com/naturegenetics the RT–PCR product contain polymor- type? Yes! The full knowledge of the risk 13. Cowles, C.R., Hirschhorn, J.N., Altshuler, D. & Lander, E.S. Nat. Genet. 32, 432–437 (2002). phisms, which is not possible for variant is important, as it helps refine the 14. Hardison, R.C., Oeltjen, J. & Miller, W. Genome Res. (such as TNF) that lack exonic SNPs. By knowledge of gene transcription and points 7, 959–966 (1997).

A clinician’s plea

Judith G. Hall © Group 2003 Nature Publishing Department of Pediatrics and Medical Genetics, University of British Columbia, Vancouver, British Columbia V6H 3V4, Canada. e-mail: [email protected]

For the detection of human gene mutations to have practical application, clear clinical descriptions of the affected individuals (as well as those clinically affected in whom mutations are not found) should be part of the publication.

Over the last ten years, there has been an in part by the promise that insights into on page 459, or what additional features exponential increase in the number of human health and disease would result. might have been present in the 31 individu- papers describing mutations in genes The large number of excellent articles als with anophthalmia who had no muta- responsible for human disease. Clinicians describing genes mutated in human dis- tions in SOX2, as outlined by Judy Fantes are thrilled that this new information may eases in this issue of Nature Genetics1–8 and colleagues3 on page 461. Did they have translate into meaningful changes in reflects the fruits (and hard work) that are other structural or functional abnormali- health care for affected individuals. We to be celebrated. At the same time, how- ties? What was different about the pregnan- carefully read such papers for clinical ever, they show how little we know about cies of those individuals? Has unusual details to help determine which individu- these genes, how much more work there is behavior developed as they age? Was their als and which families might benefit from to be done and how urgently accurate and mother’s health or nutrition unusual before molecular evaluation or testing. Some- clear clinical descriptions are needed. conception or during the pregnancy? times these details are there, but more Often we read that the ‘found’ gene is often they are not. Thus, clinical geneti- The clinical approach actually one that was previously unknown. cists are frustrated by an inability to deter- A clinician can’t help but wonder what was The gene structure may allow speculation mine to which condition or conditions the different about the 11 individuals with about function, and there may be related reported gene mutation actually refers. Ondine’s curse (congenital central genes that suggest clues as to pathway or The project to sequence the human hypoventilation syndrome) in whom no pathogenesis, but the actual pathogenic genome, nearing completion this year, has mutation in PHOX2B was found, as mechanism must be determined through been a remarkable effort. It was powered described by Jeanne Amiel and colleagues1 the hard work of defining time-specific, tis-

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sue-specific and species-specific expression because the clinician is dealing with details.” Without clear clinical descriptions, and through building models of dis- human beings, there are many constraints however, the relevance for human disease, ease. The isoforms, protein folding, control and diagnosis and therapy are the goals. diagnosis and therapy may be lost—and, mechanisms and position along a biochem- The clinician needs clinical details and after all, that is part of the purpose of such ical pathway await elucidation. clues from the affected individual, the fam- work. The clinician usually has a system- Important clues will probably come from ily and a literature review to determine atized way of collecting information (see full clinical descriptions of which tissues are which tests are appropriate to make a diag- box) and storing it for future reference involved, when the onset of disease occurs nosis. Gender, ethnic background, age and (medical records). Some papers that report and what complications develop over time. natural history help in that process. For mutations in a human gene include tables Recognizing the signs and symptoms that example, for individuals with small heads giving phenotypic features2,3,7,8. But too identify exceptions should give hints about (microcephaly), when should Seckel syn- few papers include the clinical features of alternative pathways. For instance, as indi- drome (see the paper by Mark O’Driscoll those individuals in whom no mutation cated by Catherine Dodé and colleagues2 on and colleagues5 on page 497) be consid- was found. But what is or was different page 463, among the individuals with Kall- ered? Historically, individuals with Seckel about those individuals? Occasionally, mann syndrome who have mutations in syndrome have been described as having DNA samples are obtained from cell reposi- FGFR1 (but not among those with other intrauterine growth retardation (small at tories that are notorious for lacking good, mutations), marked craniofacial structural birth for their gestational age) and a head verifiable clinical information—yet another anomalies are often seen. Those individuals size that is much smaller than would be problem! As genomic research moves with Smith–Magenis syndrome who have expected. There are many conditions that towards proteomics, and proteomics moves heart and renal defects are likely to have share these two features9. Thus, the descrip- towards ‘proteotype–phenotype’ correla- chromosomal deletions that include RAII, tion of additional clinical features, such as tions, clarity concerning the clinical features which is described by Rebecca Slager and beaked nose, large-appearing eyes and will become even more important. colleagues8 on page 466. sociable personality, may help the clinician Unfortunately, clinical descriptions are Traditionally, clinicians approach their to determine who should be tested for rarely static. Just as the individual changes http://www.nature.com/naturegenetics work quite differently from basic scien- mutations in ATR, which O’Driscoll et al.5 with age, so do the clinical features. The tists. Medical school provides them with a have found to be associated with Seckel syn- study of the natural history of a genetic dis- broad background in anatomy, cell biol- drome. These ‘newfangled’ DNA-based order is reflected against the normal matu- ogy, physiology, pathology and therapeu- tests are expensive (if you can even get them rational processes (physiologic changes that tics. When an affected individual or family done). Insurance companies don’t like diag- occur with aging) of human beings. How presents with a problem, the clinician col- nostic ‘fishing expeditions’. The clinicians the gene product fits into various biochem- lects historical information, carries out a tries to focus diagnostic testing and do only ical pathways will, in the long run, lead to physical examination and gathers the the most appropriate test(s) in a rational the signs and symptoms of the disease as results of laboratory and imaging studies. and justifiable manner. This requires know- normal processes go awry. Thus, recording Then, because there may be hundreds of ing the indications and the clinical features information about the natural history conditions to consider, the clinician usu- that might implicate a mutation in a spe- requires one to cast a wide net and often ally consults the literature to arrive at a cific gene. If the clinical features reported results in unexpected correlations. But that diagnosis that will allow appropriate have been unclear, the testing of appropri- is the real biology, the real secret of gene counseling and therapy. By contrast, ate individuals is unlikely to occur. function and the future challenge. © Group 2003 Nature Publishing owing to training and background, the scientist selects a problem, formulates a What information is needed? Now or never hypothesis and then does experiments, “These are meant to be molecular papers,” All research on human gene mutations collecting data to attempt to prove the you say. “Clinical information should be in requires collaboration with clinicians. The hypothesis correct or incorrect. Although a clinical journal.” “It takes too much clinicians are responsible for making the the two approaches have begun to overlap, space.” “We’re not interested in those fuzzy diagnosis, getting permission, obtaining

Clinical information that should be available on individuals reported to have disease-related gene mutations.

Although the type of information to be collected obviously will vary from disorder to disorder, the following represents a minimum standard, ideally to be defined on any person in whom a mutation is found, as well as those in whom no mutation is found. It would also be useful to record this same information about individuals who are found not to have an identifiable mutation, so that clinical distinctions can be made and appropriate testing can be done. • A full, three-generation family history with particular emphasis on the signs and symptoms of the disorder and features of pathogenetic relevance. • Natural history including other illnesses, pregnancy and birth history and a system-by-system review, including behavior, with changes over time in the areas of interest, noting the ages at which signs or symptoms develop. • Information on height, weight, head circumference, blood pressure, pulse and respirations. If the syndrome includes abnormalities of the craniofacial area, measurements of ears, eyes, head and face should be included10. If the syndrome includes disproportion, span, lower segment and measurements related to the area of disproportion should be included at various ages. A description of function, symmetry (or lack of symmetry) and longitudinal measurement during growth are desirable. •A full, system-by-system physical examination recording structural and minor anomalies. •Photographs of the face (anterior–posterior and lateral), of the whole body in underwear or bathing suit (anterior–posterior and lateral, preferably against a standard measuring instrument) and of specific features unique to the condition. •If bony changes are noted anywhere, a full skeletal survey should be recorded.

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the DNA samples and recording relevant information collected by their colleagues 3. Fantes, J. et al. Nat. Genet. 33, 461–463 (2003). clinical features. When papers describing be published or at the very least be readily 4. Haug, K. et al. Nat. Genet. 33, 527–532 (2003). 5. O’Driscoll, M., Ruiz-Perez, V.L., Woods, C.G., Jeggo, mutations in genes responsible for human accessible. Ideally, it should be part of P.A. & Goodship, J.A. Nat. Genet. 33, 467–501 diseases are published, clinical details every publication, although an accessible (2003). must be there. Experience tells us that if website is another option. If this informa- 6. Puls, I. et al. Nat. Genet. 33, 455–456 (2003). 7. Robertson, S.P. et al. Nat. Genet. 33, 487–491 (2003). the full clinical descriptions have not been tion is not available, opportunities to 8. Slager, R.E., Newton, T.L., Vlangos, C.N., Finucane, collected and recorded on the affected advance our understanding of biology and B. & Elsea, S.H. Nat. Genet. 33, 466–468 (2003). individuals whose DNA has led to identifi- human disease and to help affected indi- 9. Majewski, F., Ranke, M. & Schinzel, A. Am. J. Med. cation of a mutation, they never will be (it viduals will be lost. „ Genet. 12, 23–35 (1982). 10. Hall, J.G., Froster-Iskenius, U.G. & Allanson, J.E, is very difficult to contact people again). 1. Amiel, J. et al. Nat. Genet. 33, 459–461 (2003). Handbook of Normal Physical Measurements Authors need to insist that the clinical 2. Dodé, C. et al. Nat. Genet. 33, 463–465 (2003). (Oxford Medical Publications, Oxford, 1989).

The smoking gun of gene transfer

Gene transfers from chloroplasts to the As the authors point out, this surpris- nucleus occur naturally over evolution- ingly high frequency of gametes that ary time scales but have always been acquired a fragment of chloroplast DNA inferred from sequence comparisons, is still an underestimate for the overall never directly observed. A notable new transfer rate of bulk DNA from the http://www.nature.com/naturegenetics report has caught plastid-to-nucleus chloroplast to the nucleus, because only gene transfer in the act, providing excit- transfers of the engineered resistance ing opportunities to study its mecha- gene that resulted in proper expression nisitic details in the laboratory and its were detected. Transfers that did not ecological implications in nature. encompass the region of chloroplast DNA into which the resistance gene had About 1.5 billion years ago, a free-living been inserted also went undetected. cyanobacterium took up permanent resi- Hence, the true rate of bulk chloroplast dence in a eukaryotic host cell. The results A rare (1 in 16,000) kanamycin-resistant DNA transfer is probably higher. On the plant (photo courtesy of J. Timmis). of that symbiosis are today’s chloroplasts, other hand, N. tabacum is an allote- the photosynthesizing organelles of traploid, a circumstance that may buffer plants. One of the most important plants whose plastids—but only their plas- the potentially deleterious effects of large processes that occurred en route to the tids—were homogeneously transgenic DNA insertions in the nuclear chromo- establishment of the chloroplast as a sta- (homoplastomic transformants). Their somes, thus potentially permitting a © Group 2003 Nature Publishing bly inherited organelle (as opposed to a decisive trick was then simple: in addition to higher transfer rate than might be transient endosymbiont) was the transfer the plastid-specific resistance marker, their expected in diploids. Yet there is no evi- of genes from the cyanobacterial sym- chloroplast DNA–integrated fragment also dence one way or the other to suggest that biont’s genome to the host’s nuclear chro- contained a kanamycin-resistance gene, the chloroplast-derived fragments are mosomes. Recent estimates suggest that as which possessed a spliceosomal being inserted into active genes. Further much as 18% of the nuclear genes in Ara- under the control of promoter specific to study of the integration sites, their sur- bidopsis thaliana ultimately stem from the nuclear gene expression. These homoplas- rounding regions and the tendency of the ancestral plastid genome. But gene trans- tomic lines were used as the male (pollen) integrated DNA to undergo deletion fer from chloroplasts to the nucleus has donor in crosses to wild-type female recipi- should provide additional clues. always been indirectly inferred from gene- ents. The resulting seed was sown on The findings suggest that one in every sequence comparisons—until now. In a medium containing kanamycin. The expec- few thousand plants that we see in the recent report, chloroplast-to-nucleus gene tation was that only such seedlings would greenhouse, in cultivated fields or in transfer was observed in flagrante, captur- survive in which the kanamycin-resistance nature has a large and freshly incorpo- ing what may be nature’s most prevalent marker specific to the nuclear expression rated piece of chloroplast DNA some- kind of genetic promiscuity: lateral gene machinery had been transferred in a func- where in its nuclear that transfer from organelles to the nucleus. tional state from the chloroplast to the was integrated only one generation ago. Using experimental techniques pioneered nuclear genome. Among 250,000 progeny Given the geological time scales over by Pal Maliga, Huang et al. (Nature 422, tested, they found 16 independent which this rapid and efficient intracellular 72–76 (2003)) introduced a fragment of for- kanamycin-resistant plants (see figure) with gene transfer mechanism has been operat- eign DNA into the chloroplast genome of heritable nuclear insertions of the chloro- ing, the biggest surprise of all is perhaps tobacco, Nicotiana tabacum. The fragment, plast fragment, which in most cases segre- that there is any chloroplast-specific DNA which was stably integrated into the circular gated in a mendelian fashion. That left at all—but that is another story. „ chloroplast chromosome by homologous translates to a rate of successful chloroplast- —William Martin recombination, contained an antibiotic to-nucleus gene transfer accompanied by Institute of Botany III, University of Dusseldorf, resistance marker that is expressed in the stable nuclear expression of one in 16,000 Dusseldorf, Germany. e-mail: w.martin@ chloroplast. This allowed them to obtain pollen grains tested. uni-duesseldorf.de

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