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Rapid Telomere Shortening in Children

Rapid Telomere Shortening in Children

Telomere shortening may reflect the total number of divisions experienced by a somatic cell and is associated with replicative senescence. We found that the average rate of telomere shortening in peripheral blood mononuclear cells (PBMCs) obtained longitudinally from nine different infants during the first 3 years of life (270 bp per year) is more than fourfold higher than in adults and does not correlate with telomerase activity.

These results show that the rate of telomere loss changes during ontogeny, suggesting the existence of periods of accelerated cell division.

Because human immunodeficiency virus (HIV) preferentially infects actively dividing cells, our observation suggesting accelerated cell division in children may provide an explanation for some of the distinctive pathogenic features of the HIV disease in infants, including higher viral loads and more rapid progression to acquired immunodeficiency syndrome (AIDS).

BECAUSE TELOMERES in several different cell types1-3 shorten as a function of cell doublings in vitro and of patient age in vivo, telomere length has been proposed to be a potentially useful marker for the total number of divisions experienced by a cell and hence as a correlate of the aging process.

Telomere length may serve as a mitotic clock limiting the replicative capacity of somatic cells.4 However, most studies concerning telomere length and aging have examined telomere length in adults, particularly elderly adults.

Few studies have specifically examined telomere lengths, or other correlates of aging, in the very young, even though infancy is the stage in life when the most profound developmental changes occur and when cellular replication peaks. Molecular correlates of the aging process might be expected to show their most dramatic changes during infancy, particularly for organ systems, which display the most dramatic developmental changes during infancy, such as the immune system.

If rates of cell turnover vary during growth and development, the rates of telomere shortening should also change correspondingly. We therefore hypothesized that these changes should be greatest during the periods of life and in the tissues in which cellular replication is maximal. To explore this hypothesis and to obtain baseline values for the rate of telomere shortening in infants, we measured telomere length in the peripheral blood mononuclear cells (PBMCs) of infants.

Because the immune system undergoes such significant changes during the first years of life and because telomere lengths and the association of age with telomere length have been extensively studied in the PBMCs of adults, we determined telomere lengths from serial samples of pediatric patients over the first years of life. We found that PBMC telomeres shorten substantially faster in infants than in adults, and that infant PBMCs contain little telomerase, suggesting that rapid cell turnover may accompany the developmental processes occurring during infancy in the cell populations sampled using PBMCs.

The infant telomere shortening rates determined in this study may also serve as a baseline for comparing telomere shortening rates in patients in which PBMC turnover may be altered, for example human immunodeficiency virus (HIV) infection or congenital immunodeficiency syndromes. The implied elevation in cell turnover observed in infants may have additional ramifications for the pathogenesis of HIV disease in pediatric patients.
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MATERIALS AND METHODS
Patients and cells.
Samples from nine children were collected at multiple times during the first 3 years of life. These children were born to HIV-infected mothers and hence at risk for vertically acquired HIV infection. They were therefore closely followed with a variety of clinical and immunologic measures, including serial blood sampling (such serial blood samples would be difficult to obtain from other non-HIV exposed normal children for ethical reasons).

All of the children in this study were determined not to be infected with HIV and have had clinically normal growth and development. Samples from two adults followed longitudinally over 8 and 10 years, respectively, because they were considered to be at risk for HIV infection, but found to be repeatedly uninfected, were used for comparison. PBMCs were separated with Ficoll-Hypaque (Amersham Pharmacia, Piscataway, NJ), frozen in cryopreservative in a controlled rate freezer, and stored in liquid nitrogen.
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The studies were approved by the Institutional Review Board (IRB) at the National Cancer Institute and the IRB at the University of Medicine and Dentistry of New Jersey.
Telomere length measurements.

The telomere length assay was similar to those already described.5 In brief, high molecular weight genomic DNA was prepared (Puregene, Gentra Systems, Inc, Minneapolis, MN) and quantitated spectrophotometrically. Equal amounts of DNA (1 μg) were digested with AluI (New England Biolabs, Beverly, MA) to produce a terminal restriction fragment (TRF), an approximation of the telomere-containing DNA. Equal quantities of digested DNA were loaded on a 1% 20 × 25 cm agarose gel in 0.5X Tris-Borate-EDTA (TBE) buffer.

A pulsed-field at 6 V/cm for 20 hours at 15°C was used for the electrophoretic separation, using a pulse sequence designed to ensure good separation for sizes between 1 and 37 kb (PPI-200 Programmable Power Inverter; MJ Research, Watertown, MA). Additional precautions taken to enhance the accuracy and precision of the TRF measurements included loading three different markers (for short, 1 to 12 kb, intermediate, 4 to 23 kb, and relatively high, 10 to 50 kb, sizes) in 12 different lanes of the gel to control for region to region nonuniformities, which may diminish the accuracy of the TRF measurements.

Images of the gels stained with ethidium bromide were acquired by a quantitative imaging system constructed in our laboratory using a video camera and image analysis software to provide length calibrations and to correct for any inhomogeneities and nonuninformities across the gel. The DNA was transferred by standard Southern blotting,6 and the blotted membranes were hybridized with an alkaline phosphatase (AP)-linked telomere probe and a probe for the DNA length calibrating standards (Quick-Light Hybridization Kit; Lifecodes Corp, Stamford, CT). The blots were exposed to a chemiluminescent AP substrate, and the resulting signal was acquired with a Bio-Rad Molecular Imager (Bio-Rad, Hercules, CA) at a resolution of 0.1 mm. The acquired 16-bit images were initially quantitated using the Bio-Rad Molecular Analyst software and further analyzed with Scientist (MicroMath Scientific Software, Salt Lake City, UT).

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