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Pulmonary fibrosis: Genetic analysis of telomere‐related genes, telomere length measurement‐or both?

Pulmonary fibrosis (PF) is one of the key manifestations on the telomeropathy spectrum, a group of disorders caused by pathogenic mutations in telomerase-related genes (TRG: TERT, RTEL1, PARN, TERC, TINF2 and DKC1), identified in up to 25% of familial pulmonary fibrosis cases (FPF). Identification of germline mutations is achieved by genetic testing on DNA (with next-generation sequencing now available in many countries), while functional tests addressing telomerase activity are rarely performed. Telomere length (TL) shortens with age, and patients with TRG mutations are expected to have shorter age-adjusted TL. However, what threshold should be used to define clinically meaningful short telomeres? Historically, TL was considered pathologically reduced when <1st percentile of an age-matched control population, as seen in patients with dyskeratosis congenita, a rare and severe telomeropathy. However, TRG mutations may be associated with normal TL in patients with PF, particularly when PF is the first manifestation of a TRG mutation within a family. Indeed, in a recent hospital-based study of TL, Alder et al. reported that most TRG mutation carriers displayed TL >1st percentile and suggested that: (i) the TL varies by the affected generation, the type of mutation and the environment and (ii) the threshold that may be more likely to allow identification of TRG mutations may be higher (10th–25th) for older affected patients. TL measurement on circulating leucocytes remains difficult from a technical perspective. Flow-Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (qPCR) and Southern blot are different methods used for diagnostic purposes, but all require a well-defined control population. TL measured by FlowFISH is currently considered the gold standard in view of its very good interand intra-assay repeatability, although it is a highly specialized technique not available in routine genetic laboratories. Similarly, Southern blot from DNA is also reproducible and shows a good correlation with Flow-FISH but has limited availability and is unsuited to high-throughput testing. The use of qPCR to assess TL remains controversial. When qPCR is compared with Flow-FISH, the correlation observed between the two methods for healthy individuals and patients is poor (R2 = 0.33; R2 = 0.1). 2,3 Contradictory reports on the effect on danazol on TL in patients with telomeropathy may be related to this discrepancy. Townsley et al. found an increase in TL using qPCR in patients treated with danazol, whereas Khincha et al. did not observe increased TL in danazol-treated patients when assessed by Flow-FISH, raising the question of the most appropriate TL measurement technique. Conversely, qPCR provided reproducible data in an idiopathic pulmonary fibrosis (IPF) study, suggesting that qPCR may offer reliable results in experienced hands and under well-defined conditions, although this will require further investigation. In a recent publication in Respirology, PlanasCerezales et al. report on two interesting points: (i) TL could be analysed from buccal cells, and (ii) subtle immunological abnormalities may be predictive of shorter telomeres. In their study, the TL assessed by qPCR on DNA extracted from buccal cells had an acceptable correlation with the results of qPCRSouthern Blot from blood DNA (R2 = 0.690). They also report that low-titre antinuclear antibodies as well as other previously known factors (anaemia, thrombocytopenia or younger age) were predictive of shorter telomeres, although they used the relatively high threshold of <25th percentile to define short TL. The authors do not report on TRG mutations, and it is therefore not possible to know the predictive value of such a threshold in identifying TRG variants. Even though TL is also determined by the inherited TL and lifetime attrition, such a high threshold is more likely to be clinically useful if associated with knowledge of TRG variants. These results raise further questions. What test(s) should be done in suspected genetically determined PF: genetic analysis of TRG and/or TL measurement? Who should be genetically tested? As both results provide additive data, although both are associated with reduced overall or post-transplantation survival, ideally both analyses should be done in parallel. Several teams begin with TL measurement before genetic testing because TRG mutations are considered rare, occurring in only 1–3% of sporadic IPF patients. Indeed, Kropski et al. proposed TRG genetic testing after TL measurement and identification of shortened TL <10th percentile in clinical practice. However, two recent studies show that ‘rare’ TRG variants may not be so rare after all, even in sporadic IPF: TRG variants were identified in 149 (9%) of 1739 IPF patients included in four clinical trials (INSPIRE, CAPACITY, ASCEND and RIFF) and in 29 (11.3%) of a cohort of 262 IPF patients before lung transplantation, although a younger age than observed in usual IPF cohorts was seen in this study. Genetic testing appears indispensable to offer genetic counselling in families with FPF (pre-symptomatic and pre-natal) and to confirm the diagnosis of telomeropathy. However, interpretation of sequence variants should follow the consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Indeed, it should