# 04 - 482 Telomere Disease ## 482 Telomere Disease Parikh S et al: Diagnosis of “possible” mitochondrial disease: An exis­ tential crisis. J Med Genet 56:123, 2019. Russell OM et al: Mitochondrial diseases: Hope for the future. Cell 181:168, 2020. Saneto RP et al (eds): Mitochondrial Case Studies, Underlying Mecha­ nisms and Diagnosis. London, Academic Press/Elsevier, 2016. Wei W, Chinnery PF: Inheritance of mitochondrial DNA in humans: PART 16 Genes, the Environment, and Disease Implications for rare and common diseases. J Intern Med 287:634, 2020. Rodrigo T. Calado, Neal S. Young Telomere Disease ■ ■DEFINITION In telomere diseases (telomeropathies or telomere biology disorders), organ dysfunction is caused by excessive loss of the ends of chromo­ somes, a process termed accelerated telomere attrition, which results from germline mutations in genes involved in telomere maintenance. Inadequate repair or insufficient protection of telomeres and their resulting accelerated erosion induces cell death, deficient cell prolif­ eration, and chromosome instability; affected tissues show defective regeneration, fibrosis or replacement by fat, and a proclivity for cancer. Bone marrow, lungs, liver, and skin especially suffer accelerated telo­ mere loss and dysfunction. Telomeres shorten in humans at an average of 50 base pairs/year as measured in peripheral blood leukocytes. How­ ever, normal aging is not associated with developing manifestations of a telomere disease. In normal aging, sufficient stem cell number and function are maintained to sustain vital processes. Telomere length associates with life span in the general population. While shorter leukocyte telomeres correlate with increased mortality risk, especially from nonmalignant causes, telomere loss is not established as the cause of physiologic aging. Long telomeres due to rare inherited mutations do associate with clonal hematopoiesis and predisposition to cancer. ■ ■DISEASE MECHANISM Telomeres, the physical termini of linear chromosomes, are repeated hexanucleotide sequences physically associated with specific proteins. Telomeres protect the chromosome ends against recognition as damaged or infectious DNA by the DNA repair machinery (Fig. 482-1). During mitosis, the DNA polymerase employs an RNA oligonucleotide with a 3′ hydroxyl group to prime replication. The primer dissociates as the DNA polymerase advances along the template strand, and a gap is left at the ends of linear DNA molecules: the newly synthesized DNA strand is necessarily shorter than the original template—the “end-replication problem.” Chromosome erosion is thus inevitable with mitotic cell division, but the repetitive structure buffers the loss of genetic infor­ mation. In human cells, telomeres consist of hundreds to thousands of TTAGGG tandem repeats in the leading and CCCTAA in the lag­ ging DNA strand. At birth, telomeres are long, but they inexorably shorten with aging (Fig. 482-1). In an individual cell, critically short telomere length triggers the p53 pathway, leading to proliferative arrest and apoptosis. Telomere loss is the molecular basis for the “Hayflick phenomenon,” the limit to cell division. If a cell overcomes proliferation arrest, extremely short telomeres engage the DNA dam­ age repair machinery, and chromosome end-to-end fusions, chromo­ some breaks, aneuploidy, and chromosome instability may occur. In addition to the telomere repeated sequences, a group of specialized proteins, collectively termed shelterins, directly bind to or indirectly associate with telomeres, assisting in the organization of the telomere tertiary structure and inhibiting the activity of DNA damage response proteins (Fig. 482-1). To escape telomere attrition, cells with high proliferative demand, including embryonic and adult stem cells, lymphocytes, and most cancer cells, preserve telomere length by the synthesis of telomeric repeats. Telomerase adds GTTAGG hexanucleotides to the 5′ end of the leading DNA strand using a reverse transcriptase enzyme (TERT), and TERC, its RNA template (Fig. 482-1). The telomerase holoenzyme complex comprises two copies of TERT, TERC, and dyskerin and asso­ ciated proteins. TERC binds to TERT and serves as the RNA template for its function as a reverse transcriptase. Dyskerin, encoded by DKC1, stabilizes the complex, and TCAB1, encoded by the WRAP53 gene, aids telomerase trafficking to the Cajal bodies, nuclear structures for ribonucleoprotein processing where telomerase associates with telo­ meres for elongation. Telomerase expression is tightly regulated: MYC, sex hormones, and many additional factors stimulate TERT transcrip­ tion. In mature cells, the TERT gene is highly repressed. In addition, shelterin proteins (such as POT1) regulate telomerase function and processivity (the ability to consecutively synthesize telomeric repeats in a single interaction with the telomere), modulating its catalytic activ­ ity on telomeres. Other proteins also are necessary for telomere length maintenance. RTEL1, a DNA helicase, dismantles t-loops and resolves g-quadruplexes, ensuring adequate telomere elongation. Pathologic accelerated telomere attrition has a genetic origin. Germ­ line loss-of-function mutations in genes involved in telomere biology impair telomere length repair, increasing the rate of telomere erosion in highly proliferative cells, reaching critically short lengths fast. The consequences are limited cell proliferation and impaired tissue regen­ eration. Some organs appear to be particularly susceptible to telomere erosion. Billions of blood cells are produced daily (Chap. 101), and telo­ mere attrition curtails cell proliferation, producing a hypocellular mar­ row and low blood counts. Lymphocytes also have high proliferative capacity and activate telomerase, but when telomeres are very short, B and T cells have an aged, more exhausted, and proinflammatory profile, and immunodeficiency may occur. The liver is also an organ with high proliferative capacity, and telomere dysfunction impairs hepatic regeneration after injury, with a variety of pathologic conse­ quences. The lung alveolar epithelium is in contact with exogenous toxins that stimulate regeneration, and telomere loss may cooperate with other events to hinder these physiologic responses. However, it remains unclear why other regenerative tissues, like the intestine, are less affected by telomere dysfunction, or the mechanism by which telo­ mere loss provokes a fibrotic response in the lungs (pulmonary fibro­ sis), an adipose response in the marrow (aplastic anemia), and both in the liver (hepatic steatosis and cirrhosis). These phenotypes appear to result from a combination of genetic, epigenetic, and environmental determinants. When telomeres are critically short, the DNA damage response machinery may be recruited, mistaking telomeres for damaged or infectious DNA and forcing inappropriate repair. Activation of this pathway may cause chromosome instability due to end-to-end fusion of chromosomes or translocations; these alterations generate genomic instability and potentially malignant clones of cells. That telomere dysfunction increases the risk of cancer development has been dem­ onstrated in murine models of telomerase deficiency, and patients with telomere diseases are prone to develop malignant neoplasms, particularly acute myeloid leukemia and head and neck squamous cell carcinomas. ■ ■GENETICS The pattern of inheritance may be X-linked, autosomal recessive, and autosomal dominant, and penetrance is highly variable, even within a pedigree. The genetic architecture may be complex, and affected patients may inherit pathogenic loss-of-function variants in more than one gene involved in the same telomere biology pathway. At least 17 genes have been implicated in the etiology of telomeropathies (Table 482-1). ■ ■CLINICAL MANIFESTATIONS Presentation of telomere disease in the clinic is highly variable—in the tissues affected, in the severity of organ dysfunction, and in patterns of Telomerase TERT NOP10 NHP2 RNA template Centromere TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3' AATCCCAATCCCAATCCC-5' AAUCCC Chromosome TIN2 TRF1 T loop 5' D loop A 3' Telomere length (kb) Average loss of 40–60 base pairs/year Age (years) B FIGURE 482-1  Telomeres and telomerase. A. Telomeres are ribonucleoprotein structures located at the termini of linear chromosomes inside the cell nucleus composed of hundreds of tandem hexameric DNA repeats. A group of proteins bind directly or indirectly to telomere sequences in order to provide protection to the structure and are collectively termed shelterin or telosome (TRF1, TRF2, TIN2, POT1, TPP1, and RAP1). As the 3′ end of the leading strand forms a single-stranded overhang, it folds back and invades the telomeric double helix, forming a lariat termed T loop. The telomerase complex is composed of the enzyme telomerase reverse transcriptase (TERT), its RNA component (TERC), the protein dyskerin, and associated proteins (NHP2, NOP10, and GAR). This enzymatic complex elongates telomeres by adding GTTAGG hexameric repeats to the 3′ end of the telomeric leading strand, using a sequence in TERC as the template. B. The average telomere length in human leukocytes varies: it is longer at birth (10–11 kilobases) and progressively shortens with aging (6–7 kilobases at age 90 years) at an average loss of 40–60 base pairs/year. However, there is significant variability in telomere length in each given age. disease within a pedigree and between families with similar mutations. In a same family, one individual may be severely affected, but close relatives carrying the same mutation may be asymptomatic and with normal laboratory results. Asymptomatic carriers may have subclinical organ dysfunction, which may be detected by directed or specialized testing (reduced forced vital capacity on pulmonary function test, hypocellular bone marrow at biopsy, hepatic steatosis on ultrasound). Somatic genetic rescue is a rare spontaneous genetic event in a somatic cell, conferring a selective advantage and annulling the effect of the original pathogenic germline mutation and may occur in telomere diseases, mitigating the phenotype. Clonal hematopoiesis may be mal­ adaptive, as acquired mutations in a specific set of genes (e.g., TP53) lead to myeloid neoplasms. Environmental regenerative stresses, including factors such as smoking, alcohol consumption, and viral infection, may increase sus­ ceptibility to organ damage and contribute to disease heterogeneity. Disease anticipation, in which clinical phenotype manifests at an earlier age in successive generations, is observed in some families with telomeropathies due to the direct inheritance of short telomeres in sperm and oocytes. The diagnosis of a telomere disease is suggested by personal and family history, strengthened by simple measurement of leukocyte telomere length, and usually definitively established by next-generation sequencing for genes encoding telomere repair enzyme complex and shelterin components. In a machine-learning tool that differentiates acquired immune aplastic anemia from inherited bone marrow failure syndromes, telo­ mere length is a top predictor. CHAPTER 482 GAR Telomere Disease Dyskerin Dyskeratosis Congenita  Dyskeratosis congenita is the classic telomere disease of childhood and usu­ ally diagnosed in the first two decades of life. Affected children often have at least two features of the muco­ cutaneous triad of ungual dystrophy, reticular skin pigmentation, and oral leukoplakia (Fig. 482-2). In more severe syndromes, affected newborns have cer­ ebellar hypoplasia (Hoyeraal-Hreidarsson syndrome) or exudative retinopathy (Revesz syndrome) (Fig. 482-3). Telomeres are usually extremely short, below the first percentile expected for age (Fig. 482-4). Most patients with dyskeratosis congenita develop bone marrow fail­ ure, often requiring transfusions and, ultimately, bone marrow transplant. Pulmonary fibrosis appears in as many as 20% of cases and liver disease in 10%, often after bone marrow transplant for hematopoietic failure. Other tissues and organs also may be affected (Fig. 482-3). Mutations most common in dyskeratosis congenita patients are in DKC1, TINF2, TERT, TERC, and RTEL1 genes, and triallelic inheritance (involving two genes in the same pathway) also may occur (Table 482-1). POT1 Shelterin TRF2 Rap1 TPP1 Bone Marrow Failure  Aplastic anemia (Chap. 107) is the most common major clinical manifestation of dyskeratosis congenita. However, young or older patients carrying a telomere defect, without typical physical stigmata, can also develop marrow failure. Genetic variants usually are monoallelic (one mutated allele and one wild-type allele), resulting in haploinsuf­ ficiency, and TERT, TERC, and RTEL1 are the genes usually affected. Telomere loss in these cases is often less intense than in classic dyskeratosis congenita. As a result of inadequate telomerase function, the stem cell pool is limited in size and in its ability to regener­ ate, leading to marrow hypocellularity, and insufficient production of erythrocytes, platelets, and granulocytes (Fig. 482-5). The most typical presentation is moder­ ate aplastic anemia after a long history of macrocytic mild to moderate anemia and/or thrombocytopenia, with preservation of leukocyte numbers. A comprehensive personal and family history is important, querying especially for blood count abnormalities and cytopenia as well as lung and liver disease; early hair graying, while not specific to telomeropathies, strongly suggests telomere disease in the appropriate context. Myeloid Neoplasms  Some patients diagnosed with myelodysplas­ tic syndrome (Chap. 107) or acute myeloid leukemia (Chap. 109) have a family history of bone marrow failure or other myeloid neoplasms. One of the genetic causes for myeloid neoplasia predisposition is a telomere defect, and these disorders are now classified together by the World Health Organization as “myeloid neoplasms associated with telomere biology disorders.” Telomere length measurement may be confounded by circulating immature cells, which may have very short telomeres, precluding accurate test interpretation. Pulmonary Fibrosis  Pulmonary fibrosis appears in ~20% of children with dyskeratosis congenita. Conversely, ~10–15% of patients with idiopathic pulmonary fibrosis (Chap. 304) or familial pulmonary TABLE 482-1  Genetic Variants in 13 Genes Involved in Telomere Maintenance, Inheritance Pattern, and Phenotype DYSKERATOSIS CONGENITA APLASTIC ANEMIA PULMONARY FIBROSIS CIRRHOSIS MDS/LEUKEMIA GENE Telomerase PART 16 Genes, the Environment, and Disease   DKC1 XL           TERT AD/AR AD/AR AD AD AD/AR   TERC AD/AR AD AD AD AD   NOP10 AR           NHP2 AR           WRAP53 AR         Shelterin   TINF2 AD AD AD       TERF2   AD         ACD AD         Others   RTEL1 AR AD/AR AD   AD   CTC1 AR AR         PARN     AD       USB1 AD           ZCCHC8   AD AD       NAF1     AD     Abbreviations: AD, autosomal dominant; AR, autosomal recessive; MDS, myelodysplastic syndrome; XL, X-linked. fibrosis have an etiologic telomerase gene mutation. Regardless of mutation status, most pulmonary fibrosis patients have short telomeres for their age but not as short as in dyskeratosis congenita. How telo­ mere erosion causes pulmonary fibrosis is unclear, but it might prevent adequate proliferation and regeneration of pneumocytes type II. Idio­ pathic pulmonary fibrosis due to a telomere disease usually appears after the fourth decade of life, with a restrictive pattern on pulmonary function testing associated with decreased diffusion capacity for carbon monoxide (DLCO) and a diffuse “honeycomb” appearance on high-resolution computed tomography (CT) (Fig. 482-5). Histopathol­ ogy of biopsied lung shows interstitial pneumonia. The pulmonary clinical presentation in telomere disease is indistinguishable from FIGURE 482-2  Skin manifestations of dyskeratosis congenita. The pediatric syndrome dyskeratosis congenita is characterized by the mucocutaneous triad of (A) reticular skin pigmentation, (B) oral leukoplakia, and (C, D) nail dystrophy. idiopathic pulmonary fibrosis, except that those with an underlying telomere defect may have cryptic hepatic cirrhosis, mac­ rocytosis, cytopenias, and a family history of lung, liver, or bone marrow disease. Pul­ monary arteriovenous malformation lead­ ing to right-to-left shunting is observed in patients with pulmonary fibrosis due to telomere disease. Patients with idiopathic pulmonary fibrosis or familial pulmonary fibrosis should have leukocyte telomere length assayed and, if telomeres are short, undergo screening for mutations in telo­ mere-associated genes and telomeres; telo­ mere length may be normal in some cases despite the presence of pathogenic muta­ tions. TERT, TERC, RTEL1, and PARN are the most commonly mutated genes. Liver Disease  Genetic telomere defects may cause hepatic cirrhosis (Chap. 355), nodular regenerative hyperplasia of the liver, nonalcoholic fatty liver disease (Chap. 354), and hepatocellular carcinoma (Chap. 87). Hepatocytes of most patients with cirrhosis have very short telomeres. Eroded telo­ meres limit hepatocyte proliferation, espe­ cially upon chronic injury. Additionally, hepatocytes with short telomeres display abnormal metabolic patterns and defective mitochondrial function. Abnormal liver pathology may be uncovered incidentally during the evaluation of telomeropathy patients with aplastic anemia or pulmo­ nary fibrosis, but cirrhosis also may be the sole or most prominent clinical presentation of a telomere defect. A minority of individuals with cirrhosis associated with virus B or C infection or alcohol-asso­ ciated liver disease may carry a telomere-associated gene mutation. Liver histopathology is variable, but cirrhosis is usually associated with inflammation (Fig. 482-5), increased iron deposit, positivity for CD34 in sinusoid endothelial cells, and widening of hepatocyte plates. Defec­ tive telomere maintenance may increase the susceptibility of the liver to environmental challenges, such as alcohol and viruses, increasing the risk for developing severe hepatic disease in mutation carriers. ■ ■LONG TELOMERE SYNDROME POT1 is a shelterin component that modulates telomerase access to telo­ meres, thus regulating telomere elongation. Heterozygous germline lossof-function mutations in the POT1 gene cause excessively long telomeres that clinically translate into clonal hemato­ poiesis and a predisposition to benign and malignant tumors. Very long telo­ meres appear to augment the capac­ ity for cell proliferation, facilitating the acquisition of harmful driver somatic mutations. ■ ■TELOMERE LENGTH MEASUREMENT Length of telomeres can be accurately measured in peripheral blood leuko­ cytes by commercial laboratories. Of several methods available, flow–fluo­ rescence in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (qPCR) are most widely FIGURE 482-3  Clinical consequences of telomere diseases. Telomere dysfunction affects a variety of organs: cerebellum, eyes, lungs, liver, skin, gastrointestinal tract, and the bone marrow. utilized. Both methods have advantages and limitations and require high-quality samples, usually fresh or freshly processed, as cell death and DNA degradation impact the accuracy of testing. Results are usu­ ally expressed as leukocyte telomere length in kilobases. However, the interpretation of length must account for patient age, due to physi­ ologic telomere loss. A normal range for telomeres is available for each year of age, longest at birth and shortening at 40–60 base pairs per year (Fig. 482-1). For each age bracket, the percentile curves are calculated, and a given patient’s test result is interpreted in the context of normal age variation: telomeres below the tenth percentile for age are defined as “short” and telomeres below the first percentile are considered “very short” (Fig. 482-4). Telomeres above the 99th percentile are considered “very long.” Short telomeres may also be present in some chronic conditions, such as cardiovascular disease or diabetes. In these settings, telomere Telomere Diseases Dyskeratosis congenita Aplastic anemia Pulmonary fibrosis Long telomere syndrome Telomere length (kb) Age (years) FIGURE 482-4  Telomere length measurement in telomere diseases. Telomeres shorten with aging, and solid curves represent the percentiles for age in healthy subjects. Telomeres are considered “short” when below the 10th percentile, very short when below the first percentile, and very long when above the 99th percentile. In patients with dyskeratosis congenita, telomeres are usually below the first percentile, regardless of the gene lesion, whereas in patients with aplastic anemia or pulmonary fibrosis, telomeres are usually below the 10th percentile. In patients with long telomere syndrome, telomeres are usually above the 99th percentile. CHAPTER 482 Telomere Disease erosion is not thought to be etiologic but rather a secondary conse­ quence of chronic inflammation; telomere testing does not have clini­ cal utility and is not recommended. Likewise, telomere length tests, despite commercial hyperbole, have not been shown to be useful in the assessment of aging and longevity or as a basis for therapeutic interven­ tions, absent a diagnosis of genetic telomere disease. Flow-FISH uses a fluorescent-labeled nucleotide probe specific for telomere repeats to estimate telomere content in an individual cell. It has the advantage of determining telomere length in individual cells and in leukocyte subpopulations; lymphocyte telomere shortening is more specific for telomere diseases than in other cells. A limitation of flow-FISH is the requirement for intact cells for analysis, which are not always available, and neutrophils are susceptible to damage during processing, freezing, and thawing. qPCR uses telomere-binding modified primers to measure telomere content in comparison to a housekeeping gene in the whole leukocyte population and thus does not require intact cells. qPCR provides an estimate of the average telomere length of a given sample without determining telomere length in individual cells. Good DNA quality is essential for adequate qPCR testing and automation or semi-automation is required for clinical purposes, as variability in conditions among batches may result in interassay variation. The standard Southern blot is very accurate but requires larger amounts of DNA and is labor intensive. Other measures have been developed in research laboratories (single telomere length analysis [STELA], telomere shortest length assay [TESLA]) to assess critically short telomeres in particular. 10% 50% 90% 99% ■ ■GENETIC TESTING 1% When a patient with a suspected telomeropathy has short or very short telomeres, genetic screening for mutations in genes involved in telomere maintenance and biology is indicated (Table 482-1). Genetic testing has been restricted to patients with suspected telomere disorders but is increasingly incorporated into genomic screening in the bone marrow failure syndromes in general, and next-generation sequencing has been routinely used. Mutations may be biallelic or trial­ lelic involving two genes (especially in dyskeratosis congenita), but usually only one allele is mutated. Interpretation of genetic screening results is challenging, as rare singleton polymorphisms of unknown significance have been identified in large cohorts of healthy individu­ als. In silico analysis, mutation location, and functional studies are helpful to interpret the mutation significance.