The Gene

by Siddhartha Mukherjee

  Leonore died on May 14, 1978. Seventeen months later, in October 1979, Nancy attended a genetics workshop in Washington where she heard about Botstein’s gene-mapping technique. The method was still largely theoretical—thus far, no human gene had been successfully mapped with it—and the likelihood of using the method to map the Huntington’s gene was remote. Botstein’s technique was, after all, crucially dependent on the association between a disease and markers: the more patients, the stronger the association, the more refined the genetic map. Huntington’s chorea—with only a few thousand patients scattered across the United States—seemed perfectly mismatched to this gene-mapping technique.

  Yet Nancy Wexler could not shake the image of gene maps from her mind. A few years earlier, Milton Wexler had heard from a Venezuelan neurologist of two neighboring villages, Barranquitas and Lagunetas, on the shores of Lake Maracaibo in Venezuela with a striking prevalence of Huntington’s disease. In a fuzzy, black-and-white home movie filmed by the neurologist, Milton Wexler had seen more than a dozen villagers wandering woozily on the streets, their limbs shaking uncontrollably. There were scores of Huntington’s patients in the village. If Botstein’s technique had any chance of working, Nancy Wexler reasoned, she would need to access the genomes of the Venezuelan cohort. It was in Barranquitas, several thousand miles from Los Angeles, that the gene for her family’s illness would most likely be found.

  In the winter of 1979, Wexler set off to Venezuela to hunt the Huntington’s gene. “There have been a few times in my life when I felt certain that something was really right, times when I couldn’t sit still,” Wexler wrote.

  At first glance, a visitor to Barranquitas might notice nothing unusual about its inhabitants. A man walks by on a dusty road, followed by a band of shirtless children. A thin, dark-haired woman in a floral dress appears from a tin-roof shed and makes her way to the market. Two men sit across from each other, conversing and playing cards.

  The initial impression of normalcy changes quickly. There is something in the man’s walk that seems profoundly unnatural. A few steps, and his body begins to move with jerking, staccato gestures, while his hand traces sinuous arcs in midair. He twitches and lunges sideways, then corrects himself. Occasionally, his facial muscles contort into a frown. The woman’s hands also twist and writhe, tracing airy half circles around her body. She looks emaciated and drools. She has progressive dementia. One of the two men in conversation flings out his arm violently, then the talk resumes, as if nothing has happened.

  When the Venezuelan neurologist Américo Negrette first arrived in Barranquitas in the 1950s, he thought that he had stumbled upon a village of alcoholics. He soon realized that he was wrong: all the men and women with dementia, facial twitches, muscle wasting, and uncontrolled movement had a heritable neurological syndrome, Huntington’s disease. In the United States, the syndrome is fleetingly rare—only one in ten thousand have the disease. In some parts of Barranquitas and nearby Lagunetas, in contrast, nearly one in ten or one in twenty men and women were afflicted or were carriers of the gene—about two thousand in total. All have descended from a common ancestor, a woman named María Concepción—a strangely apt name—who conceived the first family that carried the mutant gene to these villages in the nineteenth century. The disease was so ubiquitous that the locals simply called it el mal—the evil.

  Wexler landed in Maracaibo in December 1979. She hired a team of eight local workers, ventured into the barrios along the lake, and began to document the pedigrees of affected and unaffected men and women (although trained as a clinical psychologist, Wexler had, by then, become one of the world’s leading experts on choreas and neurodegenerative illnesses). “It was an impossible place to conduct research,” her assistant recalled. A makeshift ambulatory clinic was set up so that neurologists could identify the patients, characterize the disease, and provide information and supportive care. At first, Wexler’s team struggled to find patients with Huntington’s disease: the illness was clustered in certain families in certain fishing villages located deep in the swamps. Then, one morning, a local fisherman brought a crucial clue: he knew of a boating shanty, about two hours along the lake, where several families were afflicted by el mal. Would Wexler like to venture through the swamps to the village?

  She would. The next day, Wexler and two assistants set off on a boat toward the pueblo de agua, the village on stilts. The heat was sweltering. They paddled for hours through the backwaters—and then, as they rounded the bend of an inlet, they saw a woman with a brown-print dress sitting cross-legged on a porch. The arrival of the boat startled the woman. She rose to go inside the house and was suddenly struck, midway, by the jerking choretic movements characteristic of Huntington’s disease. A continent away from home, Wexler had come face-to-face with that achingly recognizable dance. “It was a clash of total bizarreness and total familiarity,” she recalled. “I felt connected and alienated. I was overcome.”

  Moments later, as Wexler paddled into the heart of the village, she found another couple lying on a hammock, shaking and dancing violently. There were fourteen children, some affected and some carriers. As Wexler collected information about the children and their children, the documented lineage grew rapidly. In a few months, she had established a list containing hundreds of men, women, and children with Huntington’s disease. Over the next months, Wexler returned to the sprawl of villages with a team of trained nurses and physicians to collect vial upon vial of blood. The blood was then shipped to the laboratory of James Gusella, at the Massachusetts General Hospital in Boston, and to Michael Conneally, a medical geneticist at Indiana University.

  In Boston, Gusella purified DNA from blood cells and cut it with a barrage of enzymes, looking for a variant that might be genetically linked to Huntington’s disease. Conneally’s group analyzed the data to quantify the statistical link between the DNA variants and the disease. The three-part team expected to plod along slowly—they had to sift through thousands of polymorphic variants—but they were immediately surprised. In 1983, barely three years after the blood had arrived, Gusella’s team stumbled on a single piece of variant DNA, located on a stretch of chromosome four, that was strikingly associated with the disease. Notably, Gusella’s group had also collected blood from a much smaller American cohort with Huntington’s disease. Here too the illness associated strongly with a DNA signpost located on chromosome four. With two independent families demonstrating such a powerful association, there could be little doubt about a genetic link.

  In August 1983, Wexler and Gusella published a paper in Nature definitively mapping the Huntington’s disease gene to a distant outpost of chromosome four—4p16.3. It was a strange region of the genome, largely barren, with a few unknown genes within it. For the team of geneticists, it was like the sudden landing of a boat on a derelict beachhead, with no known landmarks in sight.

  To map a gene to its chromosomal location using linkage analysis is to zoom in from outer space into the genetic equivalent of a large metropolitan city: it produces a vastly refined understanding of the location of the gene, but it is still a long way from identifying the gene itself. Next, the gene map is refined by identifying more linkage markers, progressively narrowing the location of a gene to smaller and yet smaller chunks of the chromosome. Districts and subdistricts whiz by; neighborhoods and blocks appear.

  The final steps are improbably laborious. The piece of the chromosome carrying the suspected culprit gene is divided into parts and subparts. Each of these parts is isolated from human cells, inserted into yeast or bacterial chromosomes to make millions of copies, and thereby cloned. These cloned pieces are sequenced and analyzed, and the sequenced fragments scanned to determine if they contain a potential gene. The process is repeated and refined, every fragment sequenced and rechecked, until a piece of the candidate gene has been identified in a single DNA fragment. The ultimate test is to sequence the gene in normal and affected patients to confirm that the fragment is altered in patients with the he
reditary illness. It is like moving door-to-door to identify a culprit.

  On a bleak February morning in 1993, James Gusella received an e-mail from his senior postdoc with a single word in it: “Bingo.” It signaled an arrival—a landing. Since 1983, when Gusella’s team had initially mapped the Huntington’s gene to chromosome four, Gusella and a team of fifty-eight scientists had spent the bleakest of decades hunting for the gene on that chromosome. They had tried all sorts of shortcuts to isolate the gene. Nothing had worked. Their initial burst of luck had run out. Frustrated, they had resorted to gene-by-gene plodding. In 1992, they had gradually zeroed in on one gene, initially named IT15—“interesting transcript 15.” It was later renamed Huntingtin.

  IT15 was found to encode an enormous protein—a biochemical behemoth containing 3,144 amino acids, larger than nearly any other protein in the human body (insulin has a mere 51 amino acids). That morning in February, Gusella’s postdoc had sequenced the IT15 gene in a cohort of normal controls and patients with Huntington’s disease. As she counted the bands in the sequencing gel, she found an obvious difference between patients and their unaffected relatives. The candidate gene had been found.

  Wexler was about to leave for yet another trip to Venezuela to collect samples when Gusella called her. She was overwhelmed. She could not stop weeping. “We’ve got it, we’ve got it,” she told an interviewer. “It’s been a long day’s journey into the night.”

  The function of the Huntingtin protein is still unknown. The normal protein is found in neurons and in testicular tissue and is required for the development of the brain. The mutation that causes the disease is even more mysterious. The normal gene sequence contains a highly repetitive sequence, CAGCAGCAGCAG . . . a molecular singsong that stretches for seventeen such repeats on average (some people have ten, while others may have up to thirty-five). The mutation found in Huntington’s patients is peculiar. Sickle-cell anemia is caused by the alteration of a single amino acid in the protein. In Huntington’s disease, the mutation is not an alteration of one amino acid or two, but an increase in the number of repeats, from less than thirty-five in the normal gene to more than forty in the mutant. The increased number of repeats lengthens the size of the Huntingtin protein. The longer protein is thought to be shredded into pieces in neurons, and these pieces accumulate in tangled spools inside cells, possibly leading to their death and dysfunction.

  The origin of this strange molecular “stutter”—the alteration of a repeat sequence—still remains a mystery. It might be an error made in gene copying. Perhaps the DNA replication enzyme adds extra CAGs to the repetitive stretches, like a child who writes an additional s while spelling Mississippi. A remarkable feature of the inheritance of Huntington’s disease is a phenomenon called “anticipation”: in families with Huntington’s disease, the number of repeats gets amplified over generations, resulting in fifty or sixty repeats in the gene (the child, having misspelled Mississippi once, keeps adding more s’s). As the repeats increase, the disease accelerates in severity and onset, affecting younger and younger members. In Venezuela, even boys and girls as young as twelve years old are now afflicted, some of them carrying strings of seventy or eighty repeats.

  Davis and Botstein’s technique of mapping genes based on their physical positions on chromosomes—later called positional cloning—marked a transformative moment in human genetics. In 1989, the technique was used to identify a gene that causes cystic fibrosis, a devastating illness that affects the lungs, pancreas, bile ducts, and intestines. Unlike the mutation that causes Huntington’s disease, which is fleetingly rare in most populations (except the unusual cluster of patients in Venezuela), the mutated variant of the cystic fibrosis is common: one in twenty-five men and women of European descent carries the mutation. Humans with a single copy of the mutant gene are largely asymptomatic. If two such asymptomatic carriers conceive a child, chances are one in four that the child will be born with both mutant genes. The consequence of inheriting two mutant copies of the CF gene can be fatal. Some of the mutations have a nearly 100 percent penetrance. Until the 1980s, the average life span of a child carrying two such mutant alleles was twenty years.

  That cystic fibrosis had something to do with salt and secretions had been suspected for centuries. In 1857, a Swiss almanac for children’s songs and games warned about the health of a child whose “brow tastes salty when kissed.” Children with the disease were known to secrete such enormous quantities of salt through their sweat glands that their sweat-drenched clothes, hung on wires to dry, would corrode the metal, like seawater. The secretions of the lung were so viscous that they blocked the airways with gobs of mucus. The phlegm-clogged airways became breeding grounds for bacteria, causing frequent, lethal pneumonias, among the most common causes of death. It was a horrific life—a body drowning in its own secretions—that often culminated in a horrific death. In 1595, a professor of anatomy at Leiden wrote of a child’s death: “Inside the pericardium, the heart was floating in a poisonous liquid, sea green in colour. Death had been caused by the pancreas which was oddly swollen. . . . The little girl was very thin, worn out by hectic fever—a fluctuating but persistent fever.” It is virtually certain that he was describing a case of cystic fibrosis.

  In 1985, Lap-Chee Tsui, a human geneticist working in Toronto, found an “anonymous marker,” one of Botstein’s DNA variants along the genome, that was linked to the mutant CF gene. The marker was quickly pinpointed on chromosome seven, but the CF gene was still lost somewhere in the genetic wilderness of that chromosome. Tsui began to hunt for the CF gene by progressively narrowing the region that might contain it. The hunt was joined by Francis Collins, a human geneticist at the University of Michigan, and by Jack Riordan, also in Toronto. Collins had made an ingenious modification to the standard gene-hunting technique. In gene mapping, one usually “walked” along a chromosome—cloning one bit, then the next, one contiguous, overlapping stretch after another. It was painstakingly laborious, like climbing a rope by placing one fist directly upon the other. Collins’s method allowed him to move up and down the chromosome with a greatly outstretched reach. He called it chromosome “jumping.”

  By the spring of 1989, Collins, Tsui, and Riordan had used chromosome jumping to narrow the gene hunt to a few candidates on chromosome seven. The task was now to sequence the genes, confirm their identity, and define the mutation that affected the function of the CF gene. On a rain-drenched evening late that summer, while both Tsui and Collins were attending a gene-mapping workshop in Bethesda, they stood penitently by a fax machine, waiting for news of the gene sequence from a postdoc researcher in Collins’s lab. As the machine spit out sheaves of paper with garbles of sequence, ATGCCGGTC . . . Collins watched the revelation materialize out of thin air: only one gene was persistently mutated in both copies in affected children, while their unaffected parents carried a single copy of the mutation.

  The CF gene codes a molecule that channels salt across cellular membranes. The most common mutation is a deletion of three bases of DNA that results in the removal, or deletion, of just one amino acid from the protein (in the language of genes, three bases of DNA encode a single amino acid). This deletion creates a dysfunctional protein that is unable to move chloride—one component of sodium chloride, i.e., common salt—across membranes. The salt in sweat cannot be absorbed back into the body, resulting in the characteristically salty sweat. Nor can the body secrete salt and water into the intestines, resulting in the abdominal symptoms.II

  The cloning of the CF gene was a landmark achievement for human geneticists. Within a few months, a diagnostic test for the mutant allele became available. By the early 1990s, carriers could be screened for the mutation, and the disease could routinely be diagnosed in utero, allowing parents to consider aborting affected fetuses, or to monitor children for early manifestations of the disease. “Carrier couples”—in which both parents happen to possess at least one copy of the mutant gene—could choose not to conceive a child, or to adop
t children. Over the last decade, the combination of targeted parental screening and fetal diagnosis has reduced the prevalence of children born with cystic fibrosis by about 30 to 40 percent in populations where the frequency of the mutant allele is the highest. In 1993, a New York hospital launched an aggressive program to screen Ashkenazi Jews for three genetic diseases, including cystic fibrosis, Gaucher’s disease, and Tay-Sachs disease (mutations in these genes are more prevalent in the Ashkenazi population). Parents could freely choose to be screened, to undergo amniocentesis for prenatal diagnosis, and to terminate a pregnancy if the fetus was found to be affected. Since the launch of the program, not a single baby with any of these genetic diseases has been born at that hospital.

  It is important to conceptualize the transformation in genetics that occurred between 1971—the year that Berg and Jackson created the first molecule of recombinant DNA—and 1993, the year that the Huntington’s disease gene was definitively isolated. Even though DNA had been identified as the “master molecule” of genetics by the late 1950s, no means then existed to sequence, synthesize, alter, or manipulate it. Aside from a few notable exceptions, the genetic basis of human disease was largely unknown. Only a few human diseases—sickle-cell anemia, thalassemia, and hemophilia B—had been definitively mapped to their causal genes. The only human genetic interventions available clinically were amniocentesis and abortion. Insulin and clotting factors were being isolated from pig organs and human blood; no medicine had been created by genetic engineering. A human gene had never intentionally been expressed outside a human cell. The prospect of changing an organism’s genome by introducing foreign genes, or by deliberately mutating its native genes, was far outside the reach of any technology. The word biotechnology did not exist in the Oxford dictionary.