Dan E. Arking*, Alice Krebsova [dagger] , Milan Macek Sr. [dagger] , Milan Macek Jr. [dagger] , Albert Arking [Dagger] , I. Saira Mian***, Linda Fried, Ada Hamosh*, Srabani Dey*, Iain McIntosh*, and Harry C. Dietz*, [||] ,** "Association of Human Aging with a Functional Variant of Klotho," Proc. Natl. Acad. Sci. USA, Vol. 10,.p. 1073, Published online before print (January 15, 2002).

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* Institute of Genetic Medicine,
[||] Howard Hughes Medical Institute, and
 Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205;
[dagger] Institute of Biology and Medical Genetics, Department of Molecular Genetics-Cystic Fibrosis Center and Center for Integrated Genomics, University Hospital Motol and 2nd School of Medicine, Charles University, Prague, Czech Republic;
[Dagger] Johns Hopkins University, Baltimore, MD 21218; and
*** Department of Cell and Molecular Biology (MS 74-197), Life Science Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720

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** To whom reprint requests should be addressed at: Johns Hopkins School of Medicine, 720 Rutland Avenue, Ross Building, Room 858, Baltimore, MD 21205.
E-mail: hdietz@ihmi.edu .

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Edited by William S. Sly, St. Louis University School of Medicine, St. Louis, MO, and approved November 21, 2001 (received for review September 13, 2001).

ABSTRACT:

Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether variation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite markers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity, Bohemian Czechs, we demonstrated significant differences in selected marker allele frequencies between newborn and elderly individuals (P < 0.05). These results precipitated a search for functional variants of klotho. We identified an allele, termed KL-VS, containing six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S. Homozygous elderly individuals were underrepresented in three distinct populations: Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans [combined odds ratio (OR) = 2.59, P < 0.0023]. In a transient transfection assay, secreted levels of klotho harboring V352 are reduced 6-fold, whereas extracellular levels of the S370 form are increased 2.9-fold. The V352/S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare example of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally important. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-[beta]-D-glucoside. These results suggest that the KL-VS allele influences the trafficking and catalytic activity of klotho, and that variation in klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.

Gene May Cut Human Life Short

January 14, 2002; Washington, D.C. ( Reuters) -- A gene named after one of the Greek Fates seems to indeed hold a person's life in the balance, cutting short one's allotted time on this planet, researchers said Monday. One version of the gene, called klotho, is much more common in newborns than in 65-year-olds, which suggests it does something to reduce lifespan, said the team at Johns Hopkins University in Baltimore, MD, working with a group in Czechoslovakia.

"It seems that carrying two copies of this gene is detrimental to survival," Dan Arking, who is studying human genetics and who led the work, said in a telephone interview. "We found that infants that have two copies of the variant, one from each parent, have a frequency of about 3 percent in the populations we looked at." Only 1.1 percent of the people over 65 they checked carried two copies of the changed gene, the researchers report in Tuesday's issue of the Proceedings of the National Academy of Sciences. "So those people are dying off," Arking said.

Klotho was one of the three Greek Fates -- goddesses who in Greek mythology controlled a person's destiny and life. Klotho, whose name means " spinner," spun the thread of life. Lachesis measured the thread and Atropos cut it.

The Hopkins team looked at the genes of more than 2,000 ethnically distinct people -- 611 infants and 435 elderly Bohemian Czechs over the age of 75, a mixed group of central Europeans, and people of both European and African descent living in the Baltimore area. "What's so striking about the klotho variant is that it is relatively common and has its effect by age 65," Arking said. "About a quarter of the population had one variant copy, making them carriers." The researchers do not know what the gene does. It is not associated with any disease -- yet.

Arking said his team was building on work done by Japanese scientists, who created mice that did not have any klotho gene. "They showed mice with deficient klotho age prematurely," Arking said. "What was intriguing was that they aged like humans. They get atherosclerosis, osteoporosis, emphysema -- these are characteristics of aging that are not normally associated with mice." So his team looked for klotho in people.

There is a lot more work to be done, Arking stressed, before it will be useful to know whether a person has the "early death" version of klotho. "We can't distinguish at what stage these people are dying out," he said. "We don't know if people are dying at age 20, age 40, or age 2." Nor, he said, "is it clear what is causing their deaths." "Having one copy of the gene may be a good thing," Arking said. "For example, the gene that causes sickle cell disease when people inherit two copies seems to protect them against malaria when they get only one copy." "Perhaps the advantage can be seen only in the presence of a specific environmental stress," Arking said.