We’ll start this month’s foray into topics molecular with a reminder of some basic DNA polymerase biology. That is, they work to create one nascent strand at a time, 5’ to 3’ with respect to the growing strand and therefore 3’ to 5’ as seen along the template strand. This replication of the template strand—known as the leading strand—leaves the other template strand single stranded. Once this single stranded region is long enough, a specialized enzyme (DNA primase) lays down a short RNA primer (facing back toward the replication fork where the template strands part), and the rest of the DNA replication machinery initiates off of this and makes a new daughter strand until it bumps into, and get ligated to, the previously replicated section. This side of the fork is known as the “lagging strand,” because it’s replication lags that of the full-speed-ahead leading strand.
DNA polymerases and the terminus problem
If your chromosome is a circle, this works fine. Both strands eventually make it all the way around and close off. Many unicellular organisms, organelles likely derived from unicellular organisms (that is, mitochondria and chloroplasts), and many viruses have taken advantage of this, but large multicellular organisms like humans have linear chromosomes. (An interesting aside is why; one immediate observation is that linear chromosomes are amenable to recombination which along with sexual reproduction is a critical means of generating novel population diversity to drive adaptability. Recombination between circular chromosomes would seem to generate concatemers or other undesirable structural alterations.) Regardless of the biological reason, we’ve got linear nuclear chromosomes and we’re stuck with it. Worse, for the lagging strand at each end of a linear chromosome, this is a death sentence. Some terminal portion of that strand just won’t get replicated, and thus the intact double stranded length of the chromosome gets a little bit shorter from each end (on opposite strands) with every DNA replication cycle. While losses of 30-200 base pairs out of millions of bases pairs doesn’t sound like a lot, over biological time scales and numbers of cell divisions, it all adds up—or perhaps one should say, subtracts down—to linear chromosomes shrinking away to nothing over time, losing genes from their outer tips one by one until you’d got an inviable organism and it dies off.
Telomeres and telomerase complex
That we’re walking around proves there’s some way to rescue this problem. Actually, there’s a number of ways from polymerase priming proteins to palindromic terminal self-priming “hairpin” structures, but the one used by humans and most higher organisms is an RNA/protein complex called the telomerase holoenzyme. Its RNA component acts as a template and the proteins do various accessory functions to allow telomerase to grab onto the ends of linear chromosomes and add on multiple concatemer copies of a short DNA sequence (TTAGGG), in effect padding the ends of chromosomes with non-informational bumpers—telomeres—which can be partly lost each replication cycle without harm.
As organisms (or more properly, cell lineages within an organism, more about that later) age, telomerase tends to lose activity and so over time the length of these telomeres gets shorter and shorter. This is a normal part of cellular aging, and when telomeres (or more correctly, any one out of the 92 telomeres per cell) gets “too short,” programmed cell death (apoptosis) is triggered so the cell can be cleared away and replaced with younger, more vigorous tissue. Normally this occurs after something on the order of 50 to 70 replication cycles post fertilization for each cell lineage. (High levels of telomerase activity in gamete formation essentially “reset the clock” on egg and sperm, meaning each zygote starts this cellular timing device afresh.) Not surprisingly, this process is a topic of ongoing study as one key aspect of how organisms age as a whole.
Measuring telomeres
This discussion of long versus short telomeres suggests there must be lab methods to determine these lengths. There are a couple of methods, one based on restriction fragment sizing (which provides actual numeric length values, but is generally limited to use in research settings) and another based on quantitative PCR (qPCR; more approachable to clinical labs, but it provides a relative size against a reference in a preparation-dependent manner which is not readily amenable to comparison of results between labs). A flow cytometric approach is also possible for some cell types, as described below. In any case, measurement methods exist, and we know that in newborns telomeres are around 8 kb in length, dropping to 3 kb in adults and 1.5 kb or less in elderly people and/or rapidly dividing cell lineages. It’s noted however, that there’s quite a wide range of sizes both by age and by tissue type. Because of this wide ‘normal’ range and variance across tissues, measurement of telomere lengths in a sample is not for instance valid as a means to identify age of a forensic DNA sample source. If one were to attempt this, an additional complexity would be that data suggests an impact of genetic ethnicity on telomere length. It may however be a marker for things such as chronic inflammatory conditions, in which continuous division of immune cell populations leads to their telomeres shortening relative to less rapidly dividing tissue types in the same individual (such as skeletal muscle; heart muscle would be an even better control but is more of a challenge to obtain).
Clinical syndromes
As with any critical biochemical pathway, there are known examples of genetic diseases rooted in the telomerase system with characteristic presentations. Most serious among these is probably dyskeratosis congenita (DC), first recognized over 100 years ago and which presents as some mixture of nail dysplasia, abnormal skin pigmentation, oral leukoplakia, bone marrow failure, stenosis of various ducts (lachrymal, urethral, esophagus), liver disfunction, and a host of other problems including high incidence of several types of cancers. As of a recent review,1 underlying causes in approximately 25 percent of cases are due to mutations in the dyskerin protein (DKC1 gene, found on the X chromosome) but in the remaining cases can be traced to mutations in 13 other genes with known action in telomere maintenance. Genes on this list include TERT (the catalytic component of holoenzyme), TERC (the RNA template component), CTC1 and STN1 (along with TEN1, a trimeric modulator of telomerase activity), and RTEL1 (regulator of a required helicase activity). Because so many genes and possible mutations are involved, telomerase disorders can be observed with multiple inheritance patterns including X-linked recessive, autosomal dominant, and autosomal recessive. From a diagnostic molecular testing perspective, it is convenient that a single test—direct observation of telomere lengths (usually by fluorescent in-situ hybridization (FISH) on lymphocytes from the patient), with results scoring below one percent of population average for the patient age—is considered a reliable and specific test for this condition regardless of which underlying mutation is causal. A more detailed diagnostic follow-up would most likely be best amenable to an NGS panel approach targeted to the 14 genes referred to above.
Other named conditions closely linked to telomerase abnormalities include Hoyeraal Hreidarsson syndrome, Coats plus, and Revesz syndrome. Other conditions may be associated with telomere abnormalities but can arise from a range of other etiologies, making it unclear whether the observed telomere abnormalities are somehow causal or merely associated. Some examples of this include Myelodysplastic syndrome, fibrosis of the liver or lungs, and aplastic anemia.
Telomeres and cancer
Finally, there’s an obvious interaction between telomeres and cancer, since cancerous cells by definition among their many attributes escape senescence and become immortalized. Not surprisingly then, part of the cellular transformation process often includes reactivating or upregulating telomerase activity such that observed telomere lengths in cancer cells and cultured immortal cell lines are at the extreme upper end of what’s normally seen (around 99th percentile). There is however evidence that these telomeres may not always be normally structured and may have attributes such as significant single stranded regions which tend to lead to “sticky” chromosome ends and subsequent chromosome fusions. Such fusions and resulting breakage products are not uncommonly seen in cancerous cells. Paradoxically, while telomerase disorders result in abnormally short telomeres, cohorts of patients with one of these classical conditions show striking elevated risks to develop cancers with incidence rates as much as several hundred times that of controls.
The common activation of telomerase activity in cancer cells has led to studies on whether measurement of telomerase activity in biopsy samples can be used as a biomarker for malignancy. While this can be complicated by the fact that some normally proliferating tissue may also transiently express enough telomerase activity to be detected, some studies have shown promise in this regard although telomerase activity, in and of itself, is probably insufficient for determination of malignancy. Similarly, while it has been suggested that perhaps targeted inhibition of telomerase activity could be employed as an antineoplastic strategy, the knowledge that other non-cancerous tissues can and do express telomerase in situations such as wound healing, suggests this approach would lack specificity in targeting and likely have significant and deleterious off target effects.
In the end (no pun intended), telomeres turn out to be far more than just the aglets of our chromosomes and alterations in them can have significant clinical impacts. By nature, these are most directly ascertainable by molecular methods and thus while not the stuff of everyday diagnostics (DC has an estimated incidence rate of one to nine per 1 million births), they are a subject for analysis in at least specialty molecular lab settings and likely of interest to all.
REFERENCES
- Bertuch AA. The molecular genetics of the telomere biology disorders. RNA Biol. 2016 Aug 2;13(8):696-706.
- Savage S. Beginning at the ends: telomeres and human disease. F1000Res. 2018; 7: F1000 Faculty Rev-524.
