Introduction

The tips of human chromosomes, called telomeres, contain important genetic information and help control when cells divide. Harold C. Riethman, Ph.D., and colleagues are studying the DNA of telomeres to better understand diseases which result from damaged or rearranged telomeres. Deletion or rearrangement of small chromosome regions adjacent to telomeres (subtelomeric DNA) causes a range of disorders including mental retardation, muscular dystrophy, and heart defects. Dysfunctional telomeres are also associated with both the natural aging process and with cancer development. The Riethman lab has isolated and dissected DNA from human telomeres as part of the Human Genome Project. Molecular tools already developed as part of this effort are being used widely for detecting and characterizing subtelomeric DNA rearrangements that lead to human disease, and new tools and methods are being developed and applied to the analysis of telomere dysfunction and understanding the role of telomeres in aging and cancer.

Research Summary

Research in the Riethman laboratory focuses on analyzing the structure, function, and evolution of mammalian telomere regions.

Telomeres are dynamic and complex chromosomal structures. They are essential for genome stability and faithful chromosome replication, and mediate key biological activities including cell cycle regulation, cellular aging and immortalization, movements and localization of chromosomes within the nucleus, and transcriptional regulation of subtelomeric genes. The DNA at each human chromosome terminus is a simple repeat sequence tract (TTAGGG)_n, typically 5 kb to 15 kb in length in somatic cells, that ends with a single-stranded extension of the G-strand of DNA. The lengths of the terminal repeat tracts are dynamically modulated in a tissue-specific and individual-specific manner; loss of this sequence tract is associated with aging, and continuous maintenance of this tract is essential for cancer progression. Adjacent to this “terminal repeat” is a subtelomeric repeat region comprised of a mosaic patchwork of segmentally duplicated DNA. This class of low-copy repeat DNA is characterized by very high sequence similarity (90 % to >99.5 %) between duplicated tracts, and variably sized but often very large duplicated segment lengths (1 kb to > 200 kb). The aggregate size of a subtelomeric repeat region varies according to the specific telomere; the shortest subtelomeric repeat region is 2 kb in length and the longest is greater than 500 kb. At many individual telomeres, allelic differences in the sizes of subtelomeric repeat regions are large, on the order of hundreds of kilobases in length.

This unusual sequence organization of human telomere regions has complicated the closure phase of human genome sequencing. These same properties make subtelomere-associated regions especially prone to deletions, translocations, and other DNA rearrangements, and have led to their rapid evolution. For example, evolutionarily recent duplicative transposition of large subtelomeric DNA tracts has led to the generation of new gene families in primates, and to the formation of novel fusion transcripts with potentially new functions. In addition, instability of subtelomeric DNA leads to human diseases; the deletion of a segment of subtelomeric repeat DNA near the 4q telomere is closely linked with the genetic disease FSHD, terminal deletions of 16p result in alpha-thalassemia, and an estimated 5-10% of all cases of idiopathic mental retardation are associated with cryptic rearrangements of subtelomeric DNA. Our lab is collaboratively tracking down genes revealed by deletions and rearrangements of subtelomeric DNA that cause pediatric heart defects.

Finally, proper maintenance of the terminal (TTAGGG)_n DNA tract is critical for cell division and genome stability. Telomere shortening triggers cellular senescence or apoptosis in cells with intact checkpoint pathways. However, if these checkpoints are bypassed, continued telomere shortening leads to genome instability, crisis, and reactivation of telomere maintenance pathways which permit cellular immortalization and cancer. Patients with a form of Dyskeratosis Congenita caused by mutant telomerase RNA have both short telomeres and an increased susceptibility to malignancies, and human aging correlates both with shortened telomeres and with a dramatic rise in cancer. More generally, individuals with telomeres shorter than age-matched controls are susceptible to a range of diseases and have a higher mortality.

Our lab is developing and refining methods for accurately measuring (TTAGGG)_n tract lengths at individual human telomeres in order to investigate telomere function and dysfunction in molecular detail. In addition, this capability may reveal inborn individual-specific differences in telomere lengths that could affect susceptibility to aging and cancer.

Recent Scientific Advances

Cloning, mapping, and collaborative DNA sequencing efforts have culminated in reference sequences for each of the 41 genetically distinct human subtelomeric regions (Riethman et al. 2001, 2004). Sequence gaps that remain on the reference telomeres are generally small, well-defined, and for the most part restricted to regions directly adjacent to the terminal (TTAGGG)_n tract. Subtelomere regions are enriched 5-fold in recently-duplicated chromosome segments relative to the rest of the human genome; over half of these large DNA segments are duplicated at other telomeres. The subtelomeric sequence assemblies are also enriched > 25-fold in short, internal (TTAGGG)_n-like sequences relative to the rest of the genome; these sequence elements are involved in controlling DNA replication and in enhanced recombination in model organisms. Transcripts were annotated in each assembly; the overall transcript density is similar (within about 10%) to that which is found genome-wide, but there is wide variability in gene density among individual telomeres. Zinc finger-containing genes, olfactory receptor genes, and many additional transcript families of unknown function were found to be duplicated within and between multiple human telomere regions.

Our lab is extending this work on a subtelomeric “reference sequence” into an investigation of the extent and frequency of subtelomeric variability in humans from geographically distinct populations. Differential subtelomeric repeat content and organization at specific telomeres contribute to remarkable large-scale variations seen in human subtelomeric regions. These variations are detectable as chromosome-length polymorphisms ranging from a few kb to greater than 300 kb at a given telomere. The global complement of subtelomeric alleles in a given individual will determine the composition and dosage of functional genes embedded in the subtelomeric repeats as well as the positions of each of these genes (and the positions of adjacent 1-copy genes) relative to terminal (TTAGGG)_n tracts. Both gene dosage and gene distance from terminal (TTAGGG)_n tracts may have important consequences for expression in gene-rich subtelomeric regions, and depending upon subtelomeric gene functions and the extent of potential telomere-position effects in humans, could contribute substantially to both natural human phenotypic variation and to disease phenotypes. Most variant subtelomeric chromosome segments are not yet represented in the public sequence databases, and are therefore inaccessible for further analysis of this key chromosome region. In order to close this gap, we aim to carry out a comprehensive analysis of large-scale variations in human subtelomeric regions, to clone and collaboratively sequence subtelomeric alleles carrying unique subtelomeric-size variants at each telomere, and to develop PCR-based marker sets capable of distinguishing individual large-scale subtelomeric variants in the human population.

The newly-acquired human subtelomeric reference sequences are being used to help develop a diagnostic assay to detect subtelomeric DNA rearrangements. Current FISH-based telomeric probe sets were developed collaboratively by our lab and two others (Knight et al., 2000), and have been used widely to detect cryptic subtelomeric deletions and translocations in patients with a range of congenital disorders; this assay is currently used routinely in many clinical cytogenetic laboratories. In collaboration with a group at Children’s Hospital of Philadelphia, we are extending this work to develop a complete telomere-specific array for CGH and FISH studies of the terminal 5-10 Mb of each chromosome arm. This assay will provide simultaneous detection and precise localization of structural changes in subtelomeric DNA regions in clinical samples. The immediate application of this assay will be for collaborative detection of subtelomeric aberrations in patients with pediatric heart defects. Once established, we expect to apply the assay to studies of subtelomeric instability in human cancer.

The critical DNA regions required for developing reagents for single-telomere (TTAGGG)_n tract-length measurements are those immediately adjacent to the terminal (TTAGGG)_n tract. Within the past year we have collaboratively sequenced many of these regions in the human genome, and our ongoing projects aim to complete these sequences for all human telomeres. Several new experimental techniques for measuring single-telomere (TTAGGG)_n tract lengths are currently being explored in our lab, with the goal of producing accurate telomere-length “genotypes” of cells. This information could be used in many ways, for example in combination with subtelomeric expression profile “phenotypes” to investigate the effect of telomere lengths on subtelomeric gene expression, or in combination with epidemiological data to explore potential associations of inborn telomere-length characteristics with disease susceptibilities.