Exploring the genome treasure chest
by Susan Craig
On June 26, 2000, scientists announced they had decoded the Human Genome sequence, a working draft of a map of all the genes in the human body. And seven months later, the scientific journals Nature and Science published these working drafts. After reaching these milestones, scientists must now take the next and more important step of figuring out what genes are responsible for disease and how they work. Children's Hospital Boston researchers are among the leaders of this charge, digging through an impressive and seemingly bottomless treasure chest of information to find practical applications for the here and now.
Louis Kunkel, PhD, chief of Children's Division of Genetics, and Isaac Kohane, MD, PhD, director of Informatics, have joined forces to identify and decode genes critical to disease. Kunkel brings to the mix years of research into the gene-based disease muscular dystrophy, while Kohane brings expertise in bioinformatics, the combination of computer science and molecular biology. Together, they are unraveling the mysteries locked inside human genes.
The team is using advanced computing techniques to sift through a plethora of gene data to see which genes are turned on or off, or expressed in a particular tissue. They can then tell what a particular gene is directing cells in a specific tissue to do’Äîfor example, produce a protein for insulin or grow into a fingernail. By sifting through this data, scientists can compare and contrast expression levels, gaining information about normal gene function, as well as gene dysfunction that may be responsible for a whole host of human diseases.
Every living thing, plant or animal, is made up of cells with a set number of chromosomes where hereditary information is stored. Within each chromosome is a spiral strand of DNA that is responsible for passing genetic information from one generation to the next and for controlling cellular metabolism and protein synthesis, a vital bodily process. A gene is a section of chromosome made up of a long list of the four letters A, C, G or T, linked together in varying combinations. These letters are shorthand for the nucleotides adenine, cytosine, guanine and thymine. The Human Genome is the complete sequence of 3.5 billion of these nucleotides. There is usually a normal, or correct, "spelling" of a gene, but there can be a mistake or misspelling when the 23 pairs of chromosomes, or strings of genes, that make up a human are copied from parent to child. In this grand carbon-copy system of life, a difference in spelling may be carried down from one generation to the next.
This difference, or misspelling, called a single nucleotide polymorphism or SNP (pronounced snip), can make a gene behave differently or fail to perform its function altogether. A SNP is a substitution of one of the nucleotides in the long strain of nucleotides that make up that strand of DNA, which, in turn, makes up a single gene.
"SNPs may be misspellings or alternate spellings of a word in the storybook of a gene," says Kohane. If the Human Genome Project is thought of as a giant anthology, a particular gene can be thought of as one story in that book. The SNP, then, is a different spelling of a single word in a story.
Just as some misspellings in the English language change the meaning of a word, a modified SNP can have life-changing effects or none whatsoever. For example, if you spell "color" as "colour," the word still has the same meaning, but if you spell "where" as "wear," the word's meaning is different. In the same way, a small misspelling in the gene KCNE2 could lead a patient being treated with the common antibiotic Bactrim for a minor ear infection to have a reaction that results in permanent heart arrhythmia. Knowledge of this gene variation, before the medication is administered, could be crucial in preventing such serious side effects.
It is when this misspelling changes cellular function that disease may ensue, as is the case in patients with some types of muscular dystrophy (MD). Other times, it may not only be the SNP, but a deletion of a whole section of a gene, an entire paragraph of the story, that leads to dysfunction and disease. Indeed, this is the case with the most common forms of MD. To find a possible cure, the first challenge is to figure out whether it is a single misspelling or a missing gene that is responsible for a particular disease.
In 1986, while working in the John F. Enders Pediatric Research Laboratories at Children's, Kunkel identified the gene on chromosome X that is responsible for Duchenne muscular dystrophy. This debilitating disease, which occurs primarily in young boys, is usually fatal before its victims reach the age of 20 and claims 1,000 lives annually.
A year later, Kunkel and Children's colleague Eric Hoffman, PhD, identified dystrophin, the protein missing in patients with Duchenne muscular dystrophy. For the greater part of his professional career, Kunkel has focused his research on this protein, the genes that surround it, and their function. He defined the presence and distribution of dystrophin in the central nervous system and in recent years has furthered hope for a cure by identifying several dystrophin-associated proteins and their related disorders.
"With bioinformatics, we can take a tissue sample from a patient with muscular dystrophy, identify the dystrophin gene, measure its expression levels on a gene chip, and then compare those expressions with those of a patient who does not have MD," says Kunkel. "This allows us to look at the disease process of MD and investigate how it leads to muscle deterioration."
Children's researchers use a novel method to analyze genes in a tissue sample. This method involves the same technology that microprocessor designers use to make silicon chips. Glass slides of the genes, called microarrays or "gene chips," are made by scientists to measure gene activity. The glass slides have thousands of spots on them that represent all the genes found in DNA. The technique involves taking a blood or tissue sample and extracting its messenger ribonucleic acid or mRNA (a copy the body makes of the DNA) and placing it on the gene chip. mRNA is essentially the blueprint that tells a cell what to do with the protein it produces. A laser applies a fluorescent tag that lights up specific positions on the glass grid depending on which "blueprints" are present. The DNA and mRNAs bind, leaving a detailed grid, showing over- or under-expressed genes.
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By measuring the mRNA on the slide, scientists can tell which genes typically appear together in healthy individuals, which may be missing, and where misspellings occur in cases of dysfunction. mRNA blueprints can be likened to those of shopping malls. There are certain things that are expected from healthy genes, just as there are certain things you can count on at every shopping mall. Every mall will have a parking lot, whether that mall is in Rapid City, South Dakota, or Boston. If you are looking at genetic blueprints for possible mutations and you see that there is no mRNA for dystrophin present, for example, then there must be an error in the blueprint, like a missing parking lot. In this case, the mRNA is not able to tell the cell to produce dystrophin and the patient, therefore, is unable to rebuild muscle.
At this stage, scientists can identify the presence of the mRNA on the microarray and hypothesize what it is telling the cell to do on the protein level. The next stage, presently under way at Children's and at laboratories around the world, is exploration at the protein level, an emerging field called proteomics. Technology to measure at that precise protein level is still being developed. It is here that the genetic differences play out, leading to health or to disease.
"We measure these fluorescent spots on the microarray chip for their intensity and then use the Internet to gather more information from across the world about that gene's expression levels, and what other experiments or scientific papers have been published about that gene," Kunkel explains. "Using bioinformatics, we can find out vital information in hours or days that would have taken us months or even years to find before."
Ten years ago when a gene was discovered, the research group would publish in a scientific journal. Only after publication would others be able to build upon research done with that gene. Today, web-based databases such as UniGene, GenBank, Entrez and SGD, allow scientists to share information instantaneously. Scientists can click on dystrophin, for example, and see what other genes with which it is commonly expressed, what genes usually appear or are missing from its blueprint. Another click, and a scientist can see the latest experiments that have been done on the gene at laboratories around the world.
"When the National Institutes of Health and Celera, the private company leading the gene sequencing race, set out to decode the human genome, not even they could have forecasted the vast opportunities for scientific research that they would unleash," says Kohane.
Now, scientists all over the world, including many of those working in the John F. Enders Pediatric Research Laboratories at Children's, have opened the genome treasure chest and are unraveling the mysteries of what lies within—one gene, one SNP, one story at a time.
