by Seema Kumar
It was a clue that wasn't. When NIAMS rheumatologists, searching for the gene for familial mediterranean fever (FMF), found a family in which two of five siblings had both FMF and cystinuria -- a genetic predisposition to kidney stones -- they thought they had stumbled upon an important clue that the two diseases were genetically linked. If they found the location of the FMF gene, the researchers thought the linkage would also lead them to the gene for cystinuria. The apparent cosegregation of the two diseases in that family turned out to be a red herring; nevertheless, chasing down this clue led the researchers to successfully map the cystinuria gene on the short arm of chromosome 2. A team from Spain and Italy has found six mutations in this gene that account for cystinuria, proving that the gene causes cystinuria.
"It was a truly serendipitous situation," says Daniel Kastner, senior investigator at NIAMS, of his foray into cystinuria with Elon Pras, his postdoctoral fellow. "There is no connection whatsoever [between FMF and cystinuria]. Their occurrence in this family was just a clue that turned out to be wrong, but got us started on the pursuit of the cystinuria gene."
In fact, before they stumbled onto this family, the researchers had barely heard of cystinuria, says Kastner. "We had to first look it up [in the literature] just to find out what cystinuria was because [as] rheumatologists, we knew little beyond the fact that cystinuria causes cystine stones in the urine. That was about ... the extent of our knowledge."
The NIAMS researchers' actual objective was to find the gene for FMF, an inherited rheumatological disorder caused by a single recessive gene of unknown location and function. Stymied by the lack of families with FMF in the United States, Kastner began collaborating with the director of an FMF clinic near Tel Aviv, Israel, that follows some 2,500 FMF patients. Kastner spent the summer of 1989 in Israel collecting data from families with FMF, and his lab then began using positional cloning to map the gene for FMF. In August of 1991, Elon Pras, whose father is the director of the FMF clinic in Israel, joined Kastner's lab and "after about 10 probes, hit on the magic one on chromosome 16 that happened to be linked to the FMF gene," says Kastner. "Once we analyzed all the families, it turned out that they were all linked to chromosome 16."
In the course of collecting data on FMF, Kastner and Pras and their colleagues in Israel came across a Libyan Jewish family in which a man had married his niece and had five children, two of whom had both FMF and cystinuria. "That got us interested in this whole question of cystinuria," says Kastner.
"When you calculate the odds of cystinuria and FMF being in the same family, you get a result that suggests that the two diseases are on the same chromosome. We looked at it at that time as a good clue," says Pras.
The researchers' crash course on cystinuria taught them that the disease was one of the first disorders to be described as an inborn error of metabolism by Sir Archibald Garrod in 1908 and is, like FMF, an autosomal-recessive disease. Affected patients can develop kidney stones at any age from the first year of life; cystinuria is believed to be the most common cause of kidney stones in children and the most common single-gene hereditary cause of kidney stones. Although some patients remain asymptomatic, most have attacks of kidney stones, and in some patients, these attacks may occur as frequently as once or twice a year. The disease may run a very hard clinical course and is excruciatingly painful. Urinary obstruction can lead to colic, kidney infections, and even renal failure, and although there are treatments for the disease, cystinuria remains a significant cause of morbidity.
The researchers also learned that cystinuria is more prevalent than FMF; FMF is prevalent among North African Jews and Iraqi Jews, and among non-Jewish Armenians, but cystinuria is prevalent worldwide. Researchers estimate that 1 in 60 Americans is a carrier of the cystinuria gene and 1 in 15,000 Americans is affected by the disease. "It occurred to us ... once we had figured out the location of the FMF gene, that we could find more families with cystinuria and possibly confirm that the cystinuria gene would be near the FMF gene," says Kastner.
Soon after the team narrowed the FMF gene search to chromosome 16, Pras decided to go after the cystinuria gene while the rest of the lab continued to pursue the FMF gene. That summer, Pras found additional families with cystinuria while a new NIH clinical protocol also recruited some families with cystinuria. With a combined panel of nine families, Pras began the hunt for the cystinuria gene by looking at markers on chromosome 16, fairly confident that he would soon confirm that the gene for cystinuria was there. To their dismay, the researchers found that "even after we had looked at both ends of chromosome 16 and everywhere in between ... there wasn't a consistent linkage on chromosome 16."
The first indication that they were barking up the wrong chromosome came last spring. Pras read a crucial paper by U.S. and Spanish scientists in the Journal of Clinical Investigation reporting cloning and mapping of a human kidney gene on chromosome 2 responsible for the transport of amino acids.
"We immediately began looking at this gene as a possible candidate for the cystinuria gene," says Kastner. But when NIAMS scientists screened for chromosome 2 linkages in the nine families, they found that evidence of cystinuria linkages was only borderline: one of four affected children in one family did not fit the pattern.
Thinking that they had run into a false-positive result, the researchers decided to recruit more families. The researchers collected data from eight additional families, bringing the total number of individuals to 113, of whom 44 were affected. Results from the 17-family panel showed clearly that the gene was in a narrow area on the short arm of chromosome 2. Still, the one child did not fit the pattern.
"So we went back to that family -- an American family with both parents available and nine children, four of whom were thought to have cystinuria -- and found that the fourth affected child, a five-year-old-girl who did not fit the pattern, had been diagnosed as having cystinuria based on the fact that as an infant, she had an episode of urinary colic," says Kastner. "The cystine level in the girl's urine had never been checked, and two ultrasound tests to detect stones in her kidneys were inconclusive." The girl had been undergoing treatment, and the fact that she did not have any clinical problems was attributed to the treatment. Subsequent 24-hour urine-collection tests showed that the child did not have cystinuria. The researchers published their finding that cystinuria is linked to chromosome 2 in the April 1994 issue of Nature Genetics.
"What we have done strengthens the possibility that the transporter gene cloned by the U.S. and Spanish groups is the gene for cystinuria," says Pras. Proof that this gene does cause cystinuria came from a group of researchers from Spain and Italy who reported, in the same issue of Nature Genetics, six mutations in the gene that account for 30% of the cystinuria cases in the chromosomes they studied.
Kastner and Pras also began comparing the DNA sequences of the gene in affected and unaffected individuals in their panel of cystinuria families. Pras found mutations in one family that were different from the mutations found by the Spanish-Italian team.
The Spanish-Italian team also reported that the most common mutation, detected in three cystinuric siblings, blocked the amino acid-transport activity of the gene in Xenopus oocytes, establishing that the gene causes cystinuria. The gene normally encodes a transport protein that resorbs cystine from urine back into the blood; this process is interrupted in patients with cystinuria either because the protein is not expressed or is defective, and the excess excreted cystine precipitates, forming stones, says Pras.
"One family had gotten us interested in cystinuria, and that was just a coincidence," says Kastner. His lab has returned to the familiar territory of rheumatic diseases after this brief detour into the the realm of kidney disorders.