Multidisciplinary Collaborations Lead to Successful Genetic Research

Steps in positional cloning. Positioning of disease loci to chromosomal regions requires multidisciplinary collaborations. "You have a clinician out in Israel gathering patients, a medical geneticist doing genetic mapping, my lab doing physical mapping, and Washington University sequencing. All these things come together and, boom, in record time you have this gene," says Green. Green goes on to point out that this is a classic example of finding a gene from sequences the Human Genome Project is posting on the World Wide Web every night (www.nhgri.nih.gov/Data/#mapping). Other groups trying to find PDS were looking at the Washington University sequence as well.3 "It was a horse race. We competed to the end," says Green. Going Beyond the Gene Once researchers located PDS, they used the sequence of nucleotides to identify a protein the scientists named pendrin. A computer search found the amino acid sequence of pendrin is similar to a family of sulfate transporters studied by Lawrence Karniski, a nephrologist at the Veterans Affairs Medical Center, Iowa City, Iowa, and at the University of Iowa. Karniski was expressing sulfate transporter proteins in oocytes and baculovirus systems, so his lab had the tools and skills to do similar studies with the pendrin protein. "We spent several months trying to manipulate the system in various ways to demonstrate sulfate transport. After getting negative results, it finally dawned on us that maybe we should be looking for other possible anions the protein could transport," says Karniski. Iodide, given pendrin's high expression in the thyroid, and chloride, due to its structural similarity to iodide, were logical candidates. Karniski showed pendrin actually transports chloride and iodide ions, and not sulfate as previously assumed.4 This demonstrates the need to be careful about homology searches. Karniski warns, "Just because proteins may be similar by a computer match doesn't mean they will have the same function." Now that pendrin has been identified as a chloride-iodide transporter with a very specific and localized function in the iodide organification process, Glaser predicts, "This protein may turn out to be responsible for an aspect of basic thyroid physiology, which was unknown until now." Pendrin's role in congenital deafness is still unclear. Karniski speculates that defective chloride transport may cause abnormal fluid flux in the inner ear. Currently Karniski is expressing the mutated pendrin protein in both oocyte and baculovirus systems and trying to identify whether these mutations cause a defect in protein function, expression, or regulation. This research may shed light on how different mutations result in the different manifestations of Pendred syndrome. Most mutations cause Pendred syndrome, but a small subset appears to cause only deafness.5,6 Green notes, "It is difficult to say for certain that a given mutation only causes deafness and not thyroid disease, because there may be other contributing genetic factors." Animal models can also be developed to study Pendred syndrome's characteristic malformation of the inner ear. The National Institutes of Health is developing a knockout mouse, inactivating the mouse's normal PDS, to study how abnormal inner ear development causes deafness in Pendred syndrome. The Big Picture In the past three or four years, about 50 "nice stories" such as Pendred have surfaced, and more than 100 disease genes have been identified. Now Green predicts a major shift in human genetics: "The truth is those sorts of stories are already becoming less frequent." The first ground covered in disease-gene hunting was the identification of single-gene disorders associated with relatively common genetic diseases. That left rare genetic disorders, which are a challenge to study because of limited family resources. There may be only one or two affected families in the whole world, and often they live in remote areas with difficult access. This brings scientists to the challenge of the genetically complex common diseases such as diabetes, hypertension, heart disease, cancer, and mental illness. There are many affected individuals who exhibit the disease phenotype and can contribute genetic data. However, the disease is complicated by the interaction of multiple genes with environment. Under the right conditions each gene contributes a little bit to the inheritance of the disease. Green concludes, "Getting a handle on the genetic basis of [a condition that affects many] is critical, but untangling that mess is going to require powerful technologies and genomic information." References V.C. Sheffield et al., "Pendred syndrome maps to chromosome 7q21-34 and is caused by an intrinsic defect in thyroid iodine organification," Nature Genetics, 12:424-6, April 1996. L.A. Everett et al., "Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS)," Nature Genetics, 17:411-22, December 1997. B. Coyle et al., "Pendred syndrome (goitre and sensorineural hearing loss) maps to chromosome 7 in the region containing the nonsyndromic deafness gene DFNB4," Nature Genetics, 12:421-3, April 1996. D.A. Scott et al., "The Pendred syndrome gene encodes a chloride-iodide transport protein," Nature Genetics, 21:440-3, April 1999. X.C. Li et al., "A mutation in PDS causes non-syndromic recessive deafness," Nature Genetics, 18:215-7, March 1998. S. Usami et al., "Non-syndromic hearing loss associated with enlarged vestibular aqueduct is caused by PDS mutations," Human Genetics, 104:188-92, February 1999. The Next Frontier Genetic research is evolving rapidly as the Human Genome Project progresses. Initially, there was a rate-limiting factor of actually mapping the genes, which required finding genetic markers, pieces of DNA containing a detectable polymorphism (a DNA sequence at a chromosomal locus that varies among individuals). A genetic map is essentially a diagram that describes how known marker sequences in the chromosome separate and recombine between generations. This information can then be used to help create physical maps based on physical DNA distances measured in base pairs. Over the past 10 years some of the most accurate maps of the human genome have been created,1 and now they are being combined with family data to locate disease genes. With so much of the human genome mapped and with powerful computational tools to analyze sequences, scientists from many different disciplines can incorporate genetics into their research. Mark Boguski from the National Center for Biotechnology Information says, "Now they can do studies that were regarded as hard-core genetics only a few years ago with a minimal amount of effort." Increasingly the emphasis in genetic research is moving to gene function, as genome maps--with all the genetic material in a particular organism's cells--are being completed and becoming available to the research community. The fundamental strategy in a functional genomics approach is to systematically expand the scope of biological study from single genes or proteins to all the genes or proteins at once.2 This requires large-scale experimental methodologies combined with computational analysis of the results. Previously, computational biology would make a prediction about a protein function, and then it would take a small lab years to incrementally build up an understanding of where the protein fits into the scheme of things. The new factor in the equation is the existence of expression chips or arrays that allow the simultaneous monitoring of the expression of a large number of genes--cDNA is immobilized on glass (microarray) or synthetic oligonucleotides are immobilized on silica "chips" (probe arrays). This technology allows 10,000 different cDNA sequences to be analyzed on a 2 cm x 2 cm surface area. Though commercially available, the technology is still expensive. Large-scale analysis will not yield the intimate detail that the traditional investigations provided. However, it will rapidly narrow the gap between sequence and function to show a holistic view of biological systems. Boguski explains, "We are trying to figure out everything about a protein: every other protein it interacts with, when it gets turned on and off, what other genes get turned on and off with it." Boguski's lab is also involved in the design and construction of gene expression arrays. GenBank is a historical archive containing a tremendous amount of information, but it is not all human and there is a lot of redundancy. Therefore, computational biologists are needed to boil down the redundancy and select the best genes to put on a DNA chip. The frontier today is not computational biology alone or experimental biology alone. It is the interface between computational biology and high-throughput or large-scale experimental methodology.3 Boguski comments, "For several years we've had computer programs that can analyze the whole microbial genome overnight. We now have experimental methodologies that can analyze tens of thousands of genes overnight. Finally there's a match between the capability of computational biology and the methods that can analyze or validate the predictions." --Nadia S. Halim References P. Deloukas et al., "A physical map of 30,000 human genes," Science, 282:744-6, Oct. 23, 1998. P. Hieter, M. Boguski, "Functional genomics: it's all how you read it," Science, 278:601-2, Oct. 24, 1997. M.S. Boguski, "Bioinformatics--a new era," Trends Guide to Bioinformatics, Trends Supplement 1998, Cambridge, U.K., ier Science, pages 1-3.

Multidisciplinary Collaborations Lead to Successful Genetic Research

Going Beyond the Gene

Interested in reading more?

Become a Member of