Van Patten 8
Hannah Van Patten
BIOL 303/Sect 501
November 6, 2009
The Role of Telomerase in Autoimmune Disease, Dyskeratosis Congenita
Dyskeratosis congenita is an extremely rare hereditary, autoimmune disease that is characterized by a number of skin abnormalities. Dyskeratosis congenita (DC) results in premature aging, severe anemia due to bone marrow failure, dyskeratosis of the nails, skin hyperpigmentation or hypopigmentation, and even cancer1. The most common feature, affecting approximately 80% of DC patients, is aplastic anaemia, or bone marrow failure. For 30–50% of these cases, the patient dies prematurely due to lack of or failure of conventional treatment, whether medical therapy or bone marrow transplant2. While bone marrow failure is the leading cause of death by disease, other causes of fatality include pulmonary fibrosis and cancer3. Although the median age of diagnosis is 16, patients often die before reaching 30 years of age4. Although it only affects a small number of the world population, studying the causes of dyskeratosis congenita could provide insights into therapeutic responses to aging and cancer as well.
DC is an X-linked recessive disease caused by point mutations in the DKC1 gene. DKC1 codes for dyskerin, which is a synthase complex that mediates the posttranscriptional modification of ribosomal RNA by converting uridine to pseudouridine; dyskerin is also physically associated with the RNA component, hTR (human telomerase RNA), of human telomerase5. While DC is most commonly X-linked recessive, both autosomal dominant and autosomal recessive patterns of inheritance have also been seen. The point mutation in DKC1 that leads to X-linked recessive DC causes amino acid substitutions in the dyskerin protein complex, while the autosomal dominant form of disease arises from mutations in hTR, the locus encoding telomerase RNA6. An understanding of telomerase and its role in chromosomal maintenance and cell proliferation is essential in the study of DC.
Telomeres are non-coding, repetitive DNA sequences that serve as protective caps on the ends of chromosomes. In most somatic cells, telomeres shorten with each successive cell division until they reach a particular length, at which stage growth arrest, or senescence, occurs. If cells continue to replicate past this stage, telomeres become critically short, resulting in disruption of chromosome integrity and programmed cell death, or apoptosis7. This shortening with each division is due to incomplete lagging DNA strand synthesis and oxidative damage3. However, cells also have a mechanism of maintaining chromosome length, found in the enzyme telomerase. Telomerase is a multimeric ribonucleoprotein complex consisting of a functional RNA, hTR, that contains the template region complementary to the telomeric sequence and a reverse transcriptase protein component (hTERT) that catalyzes the addition of telomeric repeats to the ends of chromosomes (Figure 1). After dyskerin assembles with hTR, the ribosomal RNA is modified, and finally a telomere reverse transcriptase (TERT) is recruited to produce an active enzyme. While hTR is expressed in all tissues and accumulated in most somatic cells, hTERT is not, which is the reason for gradual telomere shortening. In fact, the only human cell types that activate telomerase for telomere elongation are specific germ line cells, proliferative stem cells of renewal tissues, namely epithelial and lymphoid, and immortal cancer cells6.
A study by a research team lead by Davide Ruggero sought to examine the role of the DKC1 gene in the development of DC. After generating DKC1 mutant mice, they assessed the length of telomeres of the mutant mice using flow-fluorescence in situ hybridization, or flow-FISH, to determine the molecular basis for DC. Compared to wild type mice, there were no changes in telomere length in the first three generations until a reduction of length was evident in 30% of the fourth generation. Using a telomeric repeat amplification protocol, or TRAP, assay they also showed that there was a 40% decrease in telomerase activity in the DKC1 mutant cells compared to controls. This decrease was accompanied by a 1.2-fold reduction in mouse telomerase RNA (mTR) levels. Their data suggest that although dyskerin may be an integral component of the telomerase complex, hTR especially, the DC phenotype in the early generations of mutant mice is most likely independent of its role in maintaining telomere length5. The fact that DKC1 mutant mice develop the full spectrum of DC features in G1 and G2 suggests that deregulated ribosome function is important for the initiation of DC and that impairment in telomerase activity in DKC1 mutant mice may modify or worsen the disease in later generations.
While the X-linked recessive form of DC is caused by a mutation in DKC1 gene, the autosomal dominant (AD) form of DC can be caused by several different deletion and nucleotide substitution mutations of the TERC gene, which encodes hTR3. In comparison to the X-linked DC, AD DC is a much milder form8. A study conducted at the University of Iowa by Knudsen and others examined 10 AD DC patients in the same family that all had a 74-bp deletion of the 3’ end of mature TERC RNA, as indicated by previous research. Using a TeloFISH assay to measure the average telomere length within the lymphocyte population, their results showed telomere shortening to be more prominent in the third generation family members compared to the second generation. The researchers noted that this shortened telomere length in later generations correlated with an observation of progressively more phenotypic AD DC, a phenomenon referred to as “anticipation”8. In other words, you could expect to see a disease phenotype in later-generation AD DC subjects that is more severe, appears earlier, and more closely resembles X-linked DC7. It is interesting to compare this study of a human family to the study of the mutated DKC1 in mice, especially because the X-linked DC mutation in the mice seems to show the same phenomenon of “anticipation.”
While deletion and substitution mutations in the TERC gene have both been found in AD cases of DC, AD inheritance can arise from haploinsufficiency. Haploinsufficiency signifies that the reduction in the normal level of telomerase is due to a single mutated copy of the gene, since the single functional copy of the gene cannot produce enough protein to bring about a wild-type condition3.
In a study that was published this year, one research team lead by Marie Meznikova sought to further explore the haploinsufficiency in AD DC caused by mutations of the telomerase genes, TERT or TERC. They chose mice as the test models, since mice also exhibit haploinsufficiency for the maintenance of telomeres when heterozygous for mTERT or mTERC (where m is for mouse). To determine the consequences of longterm TERT heterozygosity, they bred heterozygous mTERT animals, generated from their previous studies, with wild type C57BL/6 mice for 14 generations. Consistent with previous studies, the tenth generation heterozygous animals presented significantly shorter telomere lengths than in wild type animals. All telomere lengths were measured by Q-FISH on fixed splenocytes3. However, telomere lengths increased in generations 12-14 until the average length no longer differed significantly from that of the control mice. While this equilibration of telomere length was unexpected, it was upheld when observed in more than 27 independent crosses of non-littermates over three more years of study. They directly tested mTERT dependence of telomere lengthening by crossing a heterozygous animal with longer telomeres, generation 14, with an animal in with shorter telomeres, a third generation knockout animal from generation 12 parents. Within one generation, a statistically significant rescue of telomere signal-free ends was observed (Figure 2). Their resulting data, concurrent with previous hypotheses, established that limited telomerase activity in later generations was necessary and sufficient to rescue signal-free ends, and that lengthening segregated with the mTERT allele3.
Dyskeratosis congenita is a premature aging disorder, the effects of which can be seen on a molecular level with the rapid shortening of telomeres. This reduction in length of telomeres could be due to inheriting shorter telomeres from parents, or possibly from the reduction in telomerase activity caused by the mutations in TERT gene. Although equilibrium telomere lengths vary dramatically between yeast, mice and humans, they share a dosage-sensitive balance between telomere loss and replenishment4. This equilibrium could be very important to further research with the goal of finding a therapy in response to DC. Since mice have longer telomeres the “anticipation” phenomenon happens in even later generations. Nonetheless, the similarity between telomeric activity in mice and humans is a valuable resource for experimental data. While the levels of telomerase are essential to look at in DC patients, it would also be interesting to examine levels of telomerase and telomere lengths attributed to other symptoms of DC, including bone marrow failure and tumor growth. Further research on the effects of telomerase levels on hematopoietic cells could provide insight in other autoimmune diseases, such as rheumatoid arthritis9, in which telomerase insufficiency also plays a role.
Figure 1. Structure of telomerase complex and diseases associated with mutations in the various genes3
References
1. Nicholas, Joanne. “Mutation in DKC1 gene can cause rare aging disease and cancer.” Memorial Sloan-Kettering Cancer Center. EurekAlert. 10 Jan 2003. http://www.eurekalert.org/pub_releases/2003-01/mscc-mid010603.php- 7.5KB - Public Press Releases
2. Bailey, Penny. “Old disease, new insight: Dyskeratosis congenita: Discovery of a link between two genes responsible for a rare inherited disease, dyskeratosis congenita, is shedding light on the biology of ageing and cancer.” The Human Genome. 28 Apr 2003. < http://genome.wellcome.ac.uk/doc_WTD020832.html
3. Garcia CK, Wright WE, Shay JW. Human diseases of telomerase dysfunction: insights into tissue aging. Nucleic Acids Res. 2007;35(22):7406-16. Epub 2007 Oct 2.
4. Meznikova M, Erdmann N, Allsopp R, Harrington LA. Telomerase reverse transcriptase-dependent telomere equilibration mitigates tissue dysfunction in mTert heterozygotes. Dis Model Mech. 2009 Nov-Dec;2(11-12):620-6. Epub 2009 Oct 19.
5. Ruggero D, Grisendi S, Piazza F, Rego E, Mari F, Rao P, Cordon-Cardo C, Pandolfi PP. Dyskeratosis Congenita and Cancer in Mice Deficient in Ribosomal RNA Modification. Science 10 January 2003:Vol. 299. no. 5604, pp. 259 – 262.
6. Wong JM, Collins K. Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita. Genes Dev. 2006 Oct 15; 20(20):2848-58. Epub 2006 Oct 2.
7. Knudson M, Kulkarni S, Ballas ZK, Bessler M, Goldman F. Association of immune abnormalities with telomere shortening in autosomal-dominant dyskeratosis congenita. Blood 2005 105: 682-688. Prepublished online Jul 6, 2004
8. Vulliamy T, Marrone A, Szydlo R, et al. Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Nat Genet. 2004; 36: 447-449.
9. Fujii H, Shao L, Colmegna I, Goronzy JJ, Weyand C. Telomerase insufficiency in rheumatoid arthritis. Proc Natl Acad Sci U S A. 2009 March 17; 106(11): 4360–4365. Published online 2009 March 2.