Association of human aging with a functional variant of klotho

Dan E. Arking*, Alice Krebsova†, Milan Macek, Sr.†, Milan Macek, Jr.†, Albert Arking‡, I. Saira Mian§, Linda Fried¶, Ada Hamosh*, Srabani Dey*, Iain McIntosh*, and Harry C. Dietz*(cid:1)**

*Institute of Genetic Medicine, (cid:1)Howard Hughes Medical Institute, and ¶Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205; †Institute of Biology and Medical Genetics, Department of Molecular Genetics–Cystic Fibrosis Centre and Centre for Integrated Genomics, University Motol and 2nd School of Medicine, Charles University, Prague, Czech Republic; ‡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

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)

Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether vari- ation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite mark- ers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity, Bohemian Czechs, we demon- strated 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 identi- fied 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(cid:2)S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare ex- ample of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally im- portant. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-(cid:2)-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

to brain and testis was sufficient to rescue all features observed in klotho mice (1).

The mechanism of klotho function remains unclear. Recent experiments have shown that endothelium-dependent vasodila- tion of the aorta and arterioles was impaired in heterozygous klotho mice, but could be restored by parabiosis with wild-type mice (4). In addition, nitric oxide metabolites (NO and NO ) in 2 3 urine are significantly lower in heterozygous klotho mice (4). These results suggest that klotho protein may protect the car- diovascular system through endothelium-derived NO production (4). Remarkably, in vivo klotho gene delivery can ameliorate vascular endothelial dysfunction, increase nitric oxide produc- tion, reduce elevated blood pressure, and prevent medial hyper- trophy and perivascular fibrosis in a rat model with multiple atherogenic risk factors including hypertension, diabetes, obe- sity, and hyperlipidemia (5).

Human klotho shows 86% amino acid identity with the mouse protein, and is encoded by a gene that spans over 50 kb on chromosome 13q12 (3). To date, no premature-aging syndromes have been linked to this region. Like the mouse Klotho gene, human KLOTHO encodes both membrane-bound and secreted forms; however, in humans the secreted form predominates (3, 6). Expression of KLOTHO mRNA occurs primarily in the placenta, kidney, prostate, and small intestine (3, 6).

To determine whether the KLOTHO gene is involved in human aging, we used a population-based association study employing two newly characterized flanking microsatellite mark- ers. Marker 1 is vv.  11 kb 3(cid:2) of the last exon, and Marker 2 is   66 kb 5(cid:2) of the first exon. We present evidence that the KLOTHO locus is associated with human survival, defined as postnatal life expectancy, and longevity, defined as life expectancy after 75. We also identify and functionally characterize a variant of klotho that is associated with a decrease in survival when in homozy- gosity, in three independent populations: Bohemian Czech, Baltimore Caucasian, and Baltimore African-American.

ice homozygous for severely hypomorphic alleles of the Klotho gene (klotho mice) exhibit a syndrome resembling human aging, including atherosclerosis, osteoporosis, emphy- sema, and infertility (1). Based on both macroscopic and histo- logical appearance, klotho mice are developmentally normal until 3–4 weeks of age. Thereafter, they exhibit growth retar-   dation, gradually become inactive, and die prematurely at 8–9 weeks of age (1).

Subjects and Methods

Subjects. The Bohemian Czech subjects represent an expanded cohort from a study previously described (7). Elderly Baltimore African-American and Caucasian subjects were derived from the Women’s Health and Aging Study (8, 9), and newborn controls derived from the Baltimore–Washington Infant Study (10). Each of these studies used Institutional Research Board-approved methods of informed consent and each participant was entirely anonymous during the present study.