What is the future of telomere length testing in telomere biology disorders?

KEYWORDS:

• Telomere
• telomere biology disorder
• dyskeratosis congenita
• telomere length
• genetic testing

1. Introduction

Telomere biology disorders (TBDs) are a spectrum of genetic disorders caused by pathogenic or likely pathogenic (P/LP) rare germline variants in genes encoding essential components of telomere maintenance. Telomeres consist of long nucleotide repeats (TTAGGG)_n and a protein complex at chromosome ends essential for maintaining chromosome integrity [1]. Telomere nucleotide repeats shorten with each cell division due to the inability of DNA polymerase to fully replicate DNA ends. The reverse transcriptase, telomerase, can lengthen telomeres but is primarily expressed only in germ and stem cells. Telomere length is often considered a biomarker of aging because when telomeres reach critically short lengths cellular senescence or apoptosis is triggered.

The first link between germline defects in telomere biology genes and human disease was made in 1999 when mutations in dyskerin (encoded by DKC1) resulting in very short telomeres were identified as the cause X-linked recessive dyskeratosis congenita (DC) [2,3]. DC, the prototypic TBD, is clinically diagnosed by the presence of the mucocutaneous triad of nail dysplasia, abnormal skin pigmentation, and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure (BMF), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), head and neck squamous cell carcinoma (SCC), anogenital SCC, pulmonary fibrosis, liver disease, and many other complications. Young children with Hoyeraal-Hreidarsson syndrome, Revesz syndrome, or Coats plus have features of DC and additional other complications. Some individuals may develop only one feature of DC in middle age, such as pulmonary fibrosis or aplastic anemia [4–6].

2. Telomere length diagnostics

Accurate telomere length measurement is a key component of TBD diagnosis. Telomere restriction fragment (TRF) measurement by Southern blot is the gold standard telomere length measurement method used in research laboratories for decades [7]. However, TRF has not been used in clinical diagnostic laboratories primarily because it is very labor intensive, difficult to quantify, and requires large amounts of high-quality DNA. Relative telomere length can be determined using quantitative PCR (qPCR)but is best suited for large-scale epidemiology studies because it is not sensitive enough for diagnosing TBDs [8–10].

The diagnosis of TBDs was revolutionized with the advent of leukocyte telomere length measurement by flow cytometry with in situ hybridization (flow FISH) [11]. Lymphocyte telomere length less than the first percentile for age has proven to be highly sensitive and specific in differentiating patients with TBDs from their unaffected relatives and healthy controls and has facilitated discovery of causative genes [12]. Flow FISH can provide additional information about telomere length in hematopoiesis by measuring granulocytes, total lymphocytes, naïve T-cell, memory T-cell, B-cell, and natural killer (NK) cell telomeres. However, very few laboratories have developed expertise in flow FISH telomere length testing because it is expensive, labor intensive, and requires a fresh blood sample. US CLIA-certified laboratories currently measuring lymphocyte telomeres include Repeat Diagnostics (Vancouver, BC, Canada) and Johns Hopkins Hospital (Baltimore, MD, U.S.A) [13]. There are also centers at the University of Bern (Bern, Switzerland), Aachen University (Aachen, NRW, Germany), and the Children’s Hospital at Westmead (Westmead, NSW, Australia).

Mean flow FISH telomere length less than the first percentile for age is predictive of features associated with more severe disease such as earlier age at onset of phenotype, recessive inheritance patterns, or those with a pathogenic TINF2 variant (Figure 1) [4,14]. Adult-onset TBDs, often due to autosomal dominant inheritance, frequently show telomeres in the first to tenth percentile for age [10,13–15]. Since 10% of the general population has telomeres in this range, telomere length testing may be suggestive of disease, but additional testing is required before formal diagnosis of TBD can be rendered.

Figure 1. Telomere length and mode(s) of inheritance usually present in telomere biology disorders (TBDs). Telomere length testing should be considered for all patients with the indications shown. All patients with bone marrow failure or head/neck squamous cell carcinoma (HNSCC) should also have chromosome breakage testing of blood (and skin, if indicated) to rule out Fanconi anemia. Family history may be helpful if it is present, but many patients do not have affected relatives due to variable disease penetrance, expressivity, and/or genetic anticipation. Classic dyskeratosis congenita (DC) includes the mucocutaneous triad; Hoyeraal-Hreidarsson syndrome includes features of DC and cerebellar hypoplasia, immunodeficiency; Revesz syndrome includes exudative retinopathy, intrauterine growth restriction, intracranial calcifications as well as DC features; Coats plus has features of DC, exudative retinopathy, GI bleeding, and bone abnormalities. Figure adapted from [16].

3. Combining telomere length testing with genetic diagnosis

The importance of genetic counseling and education of patients and family members prior to telomere length and genetic testing cannot be overstated. Genetic diagnosis often has implications for the entire family as it may unexpectedly identify at risk relatives or create uncertainty if variants of uncertain significance are identified [17].

In addition to the X-linked recessive DKC1 gene, P/LP variants in at least 18 autosomal genes are associated with TBDs Figure 1. Autosomal dominant inheritance is reported with MDM4, NAF1, NOP10, NHP2, NPM1, RPA1, TERC, TINF2, TYMS, and ZCCHC8 [4–6,18]. Heterozygous TINF2 variants are often, but not always, de novo. Biallelic variants with autosomal recessive inheritance occur with CTC1, DCLRE1B, NHP2, NOP10, POT1, STN1, and WRAP53. Notably, several genes are associated with both autosomal dominant and autosomal recessive inheritance including ACD, PARN, RTEL1, and TERT. P/LP variants in these genes account for at least 80% of TBDs.

In 2023, germline genetic testing typically involves a gene panel of dozens (or more) different genes. For example, depending on the company, an inherited bone marrow failure gene panel tests for variants in 87 to 156 genes. Similarly, a pulmonary fibrosis gene panel often includes 10 to 24 genes. Most TBD-associated genes are typically included on an inherited bone marrow failure gene panel. However, a pulmonary fibrosis gene panel may only include PARN, RTEL1, TERC, and/or TERT. Newly discovered or very rare TBD-associated genes may not yet be on a gene panel. Exome sequencing and referral to a research study should be considered for patients with negative gene panel testing but high suspicion of a TBD.

Variant interpretation can be especially challenging in the TBDs because of their relative rarity and lack of robust, high-throughput functional assays. It is recommended that providers follow the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) variant curation guidelines [19] and reach out to gene- and TBD-specific experts for assistance with variant interpretation, if needed.

4. A role for new telomere length measurement methods

It is now widely recognized that it is the shortest telomere on a single chromosome arm, not the average telomere length across all chromosomes, that is responsible for triggering cellular senescence or apoptosis [20,21]. Thus, telomere length testing of the shortest telomere can serve as a marker of biological function. The extent to which the shortest telomere is associated with TBD phenotypes is just starting to be elucidated. High-throughput single telomere length analysis (HT-STELA) uses a small amount of DNA, determines the distribution of telomere lengths at 17p and XpYp telomeres, and can measure telomeres less than 3 kilobases (kb). HT-STELA successfully identified patients with TBDs, identified genotype-phenotype associations, and has recently been implemented as a diagnostic test in the United Kingdom [20]. An important strength of HT-STELA is its ability to detect ultrashort telomeres (less than 3 kb), which can potentially provide a more accurate assessment of telomere lengths than is currently measurable by flow FISH, Southern blot, or qPCR.

Although not yet clinically tested, the Telomere Shortest Length Assay (TeSLA) holds promise as a TBD diagnostic tool. TeSLA measures the lengths and tallies the numbers of single telomeres on all 23 pairs of human chromosomes, including telomeres less than 3 kb [22]. TeSLA’s major disadvantages are that it is costly and low throughput. HT-STELA and TeSLA are potentially valuable tools in disease prognostication because they detect and measure ultrashort telomeres. Longitudinal studies are required to understand the extent to which the number of ultrashort telomeres [7] is associated with clinical outcomes.

Despite STELA and TeSLA’s advantages, they are limited by their inability to detect telomeric variant sequences (TVSs), which may have implications in disease. A recently developed telomere length measurement method uses the PacBio high-fidelity mass sequencing platform which overcomes the limitations of other measurement methods [23]. However, it is far more expensive than STELA/TeSLA, requires a large amount of DNA (20 µg at minimum), and has not been systematically evaluated in. These limitations make mass sequencing less practical for diagnostic purposes now, but holds promise for the future, particularly as the role of TVS in TBDs is elucidated.

5. Can monitoring telomere length guide therapy for TBDs?

To date, the only therapeutic option that may potentially increase telomere length is androgen therapy [24-27]. Some have hypothesized that androgens lengthen telomeres by increasing telomerase activity because the TERT promoter has an estrogen response element; however, it remains unclear if this is its true mechanism as neither danazol nor its major metabolites have been shown to be capable of aromatizing to an estrogenic metabolite [28]. Though preclinical studies support that androgen treatment elongates telomeres in cell lines and mouse models, clinical studies have held more mixed results. Out of four pre- vs. post- androgen therapy studies conducted in individuals with TBDs to date, two have shown an increase in qPCR measured telomere length [25,27], one has shown an increase in flow FISH telomere length [24], and one concluded that there was no effect on flow FISH telomere length [26]. Notably, studies have been largely observational and small, consisting of seven to twelve patients with a short median follow-up of one to 5 years. The type of androgen used (e.g. danazol, oxymethalone, or nadrolone) may also have an effect but this question cannot yet be addressed with the current data.

The current state of knowledge regarding telomere length does not warrant alterations in clinical management based on longitudinal monitoring of telomere length measurements. However, it is possible that as connections between longitudinal telomere length and progression of disease are further described and new therapeutic options are discovered, rate of telomere attrition may one day guide therapeutic decision-making for TBDs.

6. What’s next?

Telomere length and germline genetic testing are interconnected modalities that have greatly facilitated TBD diagnosis and expanded understanding of TBD-associated ailments. Telomere length testing can help determine who should have genetic testing and/or help with variant interpretation in a person who has already had genetic testing.

TBD diagnostic dilemmas occur when a patient has only one feature (e.g., aplastic anemia or pulmonary fibrosis), minimal or no family history, and flow FISH telomeres in the first to tenth percentile for age. Identification of a P/LP variant in a TBD-associated gene can confirm the diagnosis in this case, although absence of a known variant doesn’t necessarily rule out a TBD as pathogenic variants in new genes are still being discovered. Flow FISH telomere lengths less than the first percentile for age in the setting of relevant clinical features can confirm a diagnosis in patients without an identified genetic variant.

Single telomere length assays, such as HT-STELA or TeSLA, are promising assays that may improve diagnosis due to increased sensitivity for ultrashort telomeres and better estimates of overall telomere length distribution in an individual.

Long read sequencing modalities hold the promise of more precision in telomere length measurement which could then be applied to the clinic. Although not yet evaluated, each of these new methods hold promise in TBD clinical prognostication. Large, well-powered clinical studies are needed to stratify individuals with telomeres in the 1^st to 10^th percentile who have a higher likelihood of having a TBD due to clinical presentation with TBD-associated phenotypical features and may benefit from subsequent genetic testing versus those with short telomeres in the general population who would likely not benefit from multigene panel testing.

Until these studies are completed, careful clinical evaluation, a detailed family history, genetic counseling, flow FISH telomere length testing, and germline genetic testing remains the optimal approach to identifying patients with TBDs and at-risk relatives.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Acknowledgments

This work was supported by the intramural research program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute. We are grateful to all patients and their families for contributing to our research. We thank Dr. Marena Niewisch for valuable discussions.

Additional information

Funding

This work was supported by the intramural research program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute.