Abstract
Background
Loss-of-function mutations in genes that encode for components of the telomere repair complex cause accelerated telomere shortening. Hepatic involvement has been recognized as a cause of morbidity in telomere diseases, but very few studies have characterized the nature and extent of liver involvement in affected patients. We report the prevalence and characteristics of liver involvement in a large cohort of patients with telomere disease evaluated serially at the National Institutes of Health.
Methods:
One hundred twenty-one patients with known or suspected telomere disease were screened; 40 patients with liver involvement were included in the current study. Median follow up was 2.4 years. Data were collected regarding their demographic information, laboratory analysis, imaging and histopathology.
Results
Forty patients (40% of the cohort) with a median age of 42 years were found to have liver involvement. Liver enzyme elevation was cholestatic in pattern; 8(21%) had drug related enzyme elevations. The most common imaging finding was increased hepatic echogenicity on ultrasound in 39% (9) patients, followed by hepatomegaly in 26% (6) subjects.
Biopsies were infrequent due to risk associated with thrombocytopenia, but in six patients there were varying findings: nodular regenerative hyperplasia, steatohepatitis, hemosiderosis, cholestasis, and cirrhosis with hepatic steatosis. Almost half the cohort had pulmonary diffusion abnormalities, and 25% died during the follow up period.
Conclusion
In patients with telomere disease, hepatic involvement is common and can present in diverse ways, including elevated liver enzymes, histopathologic as well as imaging abnormalities. Liver disease has important implications for morbidity and mortality in patients with telomere disease.
Introduction
Telomeres are repeated hexanucleotides and associated proteins found at the end of linear chromosomes. In humans, telomeres consist of hundreds to thousands of TTAGGG random repeats in the leading strand, capped by proteins called Shelterins, and provide a repetitive template for enzymatic repair of the ends of chromosomes. (1) Telomerase is a ribonucleoprotein enzyme complex comprised of a reverse transcriptase, telomerase enzyme and an RNA template with associated proteins, that ensures preservation of telomere length(2). This preservation of length however, is not complete, and telomere attrition occurs at the rate of 50–100 base pairs/cell cycle, till a critically short length on a chromosome is reached, which then triggers DNA damage responses, leading to senescence or apoptosis. (3–6).
Mutations in genes controlling telomere length as well as telomerase mutations can lead to organ dysfunction, the most common of which include pulmonary, hepatic and bone marrow abnormalities(7). Telomere disorders are frequently associated with bone marrow failure syndromes, among these, dyskeratosis congenita (DKC) is a classical telomere biology disorder, that is characterized by the clinical triad of oral leukoplakia, nail dysplasia and abnormal skin pigmentation associated with bone marrow failure. Germline mutations in several genes including the TERT, TERC, TINF2, DKC, NHP2, NOP10 and RTEL1 have been associated with the DKC phenotype.(8) Additionally, some patients with aplastic anemia, especially those with moderate decreases in hematopoietic cell lines also carry telomere mutations(9,10). Idiopathic pulmonary fibrosis (IPF) is the most commonly reported pulmonary manifestation in patients with telomere disorders(7). Eight-15% familial and 1–33% IPF is attributed to telomere mutations, including TERT, TERC, TINF2, RTEL1 and PARN.(11,12). Subjects with IPF are generally diagnosed in the fifth decade of life, but diagnosis can be delayed until as late as the ninth decade, and in some cases, liver cirrhosis is the initial presentation of telomere disease(13).
While IPF is the most common complication accounting for the majority of morbidity and mortality associated with telomere disease, liver disease has been reported in a significant number of patients.(11,13–15). Previous studies have suggested that liver disease is seen in approximately 10% of patients with telomere disease(13,16), however, these studies either report on selected syndromes associated with short telomeres, such as dyskeratosis congenita (16) and dyspnea related to pulmonary disease(15), or are based on genetic screening of families.(13)
There is a lack of understanding related to the natural history and extent of liver involvement in telomere disease. As with other rare diseases, knowledge pertaining to the hepatic manifestations of the underlying disease is important for early detection and management. We characterize liver involvement in a large cohort of patients with telomere disease, and its relationship with patient characteristics and outcomes.
Materials and methods
Patients with confirmed telomere disease followed prospectively in two clinical research studies NCT01441037 and NCT00001620 at the NIH Clinical Center were evaluated. Telomere gene sequencing was performed as previously described.(14) Subjects were considered if they had a confirmed genetic mutation in a telomere gene and/or clinical features of telomere disease and short telomeres.(11)
Out of 100 patients with known telomere disease, we evaluated 40 patients as they had findings suggestive of liver involvement, including elevated liver enzymes, abnormal imaging or liver biopsy findings. Potential liver involvement was not assessed routinely in the entire cohort.
Longitudinal demographic, clinical, radiographic and laboratory data were collected. Patient characteristics, medications, as well as other clinical events and outcomes were recorded during medical chart review. Primary medical records were reviewed by four authors (DK, RO, MHC, OKD).
Liver enzyme elevations were considered to be drug - related when a potentially hepatotoxic drug had been administered within a month of enzyme elevation (the hepatotoxic potential of a drug was identified based on drug package inserts and the Livertox database(17)). The pattern of liver enzyme elevation was classified as cholestatic or hepatocellular based on R factor calculation, where an R <2 indicates a cholestatic pattern and >5 indicates a hepatocellular pattern of liver enzyme elevation.
All subjects gave written informed consent and participated in protocols approved by the Institutional Review Board at the National Heart, Lung and Blood Institute (NHLBI).
Genetic Testing
Sequencing was performed bidirectionally with chain termination method and the BigDye Terminator version 3.1. Sequencing products were analyzed in an automated genetic-sequence analyzer. Sequence was analyzed and compared to a TERC, TERT, TERF2, TINF2 or DKC1 normal template using Sequencer software. Telomere length was defined based on leukocyte telomere lengths measured relative to age-matched normal individuals. Lengths were classified as
-
a)
very short: more than or equal to 2.6 standard deviations below the age matched mean,
-
b)
shorter: 1.2 to 1.6 standard deviations below the age matched mean,
-
c)
short: 1.7 to 2.5 standard deviations below the age matched mean and
-
d)
normal: no more than 1.4 standard deviations below the age-matched mean.
Imaging and volumetric analysis of spleen and liver
Liver imaging included liver ultrasounds as well as abdominal CT scans and MRIs. Liver and spleen sizes were measured by one of the authors (MHC) and categorized into normal and enlarged according to accepted radiologic cut-offs(18). Spleen length/height (SL/H) ratios were calculated by dividing spleen length (mm) on US by patient’s height(cm). Patients with SL/H ratios greater than upper limit of normal defined by two previously performed studies(19,20) were classified as having splenomegaly.
Liver biopsy
When clinically indicated, ie, in case of persistently elevated liver enzymes or clinical evidence suggestive of liver disease, patients were offered a liver biopsy. Liver biopsies were performed via the transjugular route due to underlying thrombocytopenia. Liver biopsies were reviewed at the NIH by a hepatopathologist (DEK).
Pulmonary studies
Pulmonary testing was performed in patients with clinical symptoms or imaging evidence of lung disease and reports were reviewed. Values of right ventricular systolic pressure (RVSP) a surrogate estimate of pulmonary artery pressure were also obtained from echocardiogram reports in patients who underwent them.
Statistical analysis
Statistical analysis was performed using Prism Graph Pad version 7 for Mac OS X, Graph Pad Software, La Jolla, CA. Summary statistics of baseline data were presented as frequencies and median with interquartile range (IQR), unless otherwise specified.
Results
Patient Characteristics
The 40 patients had a median age of 42, ranging between 13 to 73 years, 24 were males. The most common mutation was TERT in 21 patients, followed by 4 TERC and 1 TINF2 mutation. (Table 1). TERT mutation variants did not appear to cluster. (Table 4).
Table 1:
Patient characteristics
| Patient characteristics | Liver disease(n=40) |
|---|---|
| Median Age in Years (IQR) | 42(29,50) |
| Male(%) | 24(60) |
| Mutation TERC TERT DKC TINF2 Unknown |
4(10) 21(52.5) 0 1(2.5) 14(35) |
| Telomere length Normal Short Shorter Very short |
4(10) 3(7.5) 4(10) 29(72.5) |
| Pulmonary fibrosis | 8(20) |
| Deaths | 10(25) |
Table 4:
TERT gene variants in the cohort
| Subject ID | TERT gene Mutation |
|---|---|
| 1 | c.838G>A (Glu280Lys) |
| 2 | c.3184G>A (Ala1062Thr), [2389T>C (S796P)] (compound) |
| 3 | c.1931C>T (Thr644Met) |
| 4 | c.3184G>A (Ala1062Thr) |
| 5 | c.1234C>T (His412Tyr), c.3184G>A (Ala1062Thr) |
| 6 | c.1234C>T (His412Tyr) |
| 7 | c.389C>T (Ala130Val) |
| 8 | c.1234C>T (His412Tyr) |
| 9 | c.619 G>A (Val207Ile) |
| 10 | c.2665C>T (Arg889X) |
| 11 | c.1931C>T (Thr644Met) |
| 12 | c.1710G>C_T (Lys570Asn) |
| 13 | c.3026C>A (Ala1009Ile) |
| 14 | c.3184G>A (Ala1062Thr) |
| 15 | c.1211 C>G (Pro404Arg) |
| 16 | c.1603C>T (Arg535Cys) |
| 17 | c.779G>A (Gly260Asp) |
| 18 | M773T |
| 19 | 604 G/A (202 Ala/Thr) |
| 20 | c.3184G>A (Ala1062Thr) |
Biochemical pattern
39 out of 40 patients (98%) had persistent liver enzyme elevations for over six months, and one patient had abnormal hepatic imaging with normal liver enzymes. 19 patients (49%) demonstrated a cholestatic pattern of liver enzyme elevation, 8 (21%) demonstrated a hepatocellular pattern and 12 (30%) mixed pattern. (Table 2). Eight (21%) patients were thought to have drug related enzyme elevations, as they all showed improvement on withdrawing or reducing medication doses. Drugs implicated included danazol, fluconazole, cyclosporine, anti-thymocyte globulin (ATG) and valacyclovir.
Table 2:
Median laboratory parameters of patients with liver disease
| Parameter* (median, IQR) |
Liver disease(n=40) |
|---|---|
| ALT (U/L) | 76(32,122) |
| AST (U/L) | 40(29,67) |
| ALP (U/L) | 86(66,132) |
| PT(seconds) | 14.2(14.2,14.8) |
| Albumin (g/dL) | 3.5(3.2,3.7) |
| Bilirubin (direct, mg/dL) | 0.2(0.1,0.3) |
| Bilirubin (total, mg/dL) | 0.6(0.5,1) |
| Hemoglobin(g/dL) | 9(7.5,10.2) |
| Platelets (X/dL) | 23000(15000,39000) |
| Creatinine(mg/dL) | 0.8(0.62,0.93) |
median(IQR)
Imaging abnormalities
Of 40 patients studied, 23 had liver imaging performed, with a total of 40 liver ultrasounds performed for 17 patients, 40 CT scans for 16 patients, and 8 MRIs. 18 patients had both ultrasounds and CT scans performed, with similar findings. By hepatic ultrasound, the most common finding was increased liver echogenicity (n=9, 39%). Six subjects (26%) had hepatomegaly with an average liver size of 16±2.2 cm and 5 (21%) had a nodular contour suggestive of cirrhosis. Five subjects (21%) had evidence of splenomegaly, with an average spleen length of 17±4.8 cm, 2 of these also had evidence of portal hypertension on MRI (distended portal vein at 18 mm and re-canalized umbilical vein respectively). The mean age of patients with splenomegaly was 25±3 years. Five subjects (21%) had evidence of iron deposition on MRI identified by signal loss in the liver tissue. The average spleen to height ratio was 0.41±.40 mm/cm. There was no correlation of spleen by height ratios with platelet levels, ALT or AST levels. There was also no relationship of spleen size with the type of mutation present. (Splenomegaly in TERC vs TERT mutations, p=0.68).
Liver biopsy
Six of 40 (15%) patients underwent a clinically indicated liver biopsy, 5 of which were evaluated at the Clinical Center. Although more patients had clinical indications for a liver biopsy, this was limited by thrombocytopenia. Histopathologic findings varied, with nodular regenerative hyperplasia observed in one patient, steatohepatitis in three patients, of whom 2 were cirrhotic. Three patients also demonstrated significant hemosiderosis, probably as a consequence of transfusions. Only one patient, who had both cirrhosis and steatohepatitis, had normal liver enzymes at time of biopsy.
Pulmonary function
Pulmonary function tests (PFTs) were available for 21 out of 40 (52%) of the liver-affected cohort. Of these patients, according to the American Thoracic Society (ATS) guidelines(21), 6 patients (28%) were classified as restrictive ventilatory defect, 3 patients (14%) were classified as obstructive ventilatory defect, 2 patients (10%) had the nonspecific pattern, and the rest (48%) had normal spirometry and lung volumes. Regarding carbon monoxide diffusion capacity (DLCO), 19 patients (90%) had some degree of diffusion impairment. The mean DLCO adjusted for hemoglobin was 60.28%±15.8. Based on ATS guidelines, the degree of diffusion abnormality was graded as mild, moderate, and severe in 8 (38%), 9 (43%), and 2 (9.5%) patients, respectively. Only 2 patients (9.5%) had a normal DLCO.
Transthoracic echocardiography results were available for 26 out of the 40 patients (65%) with liver involvement. The right ventricular systolic pressure (RVSP), a surrogate reflector of pulmonary arterial pressure(22), could be calculated in 21 patients (81%). Elevated RVSP of 36 mm Hg or higher was documented in 3 patients (12%), while in the reminder the estimated RVSP was lower than 36 mm Hg with a mean value of 27.7±15.8 mm Hg.
Comorbidities and death
One patient required a porto-caval shunt for nodular regenerative hyperplasia complicated by portal hypertension and recurrent bleeding episodes. Of the cohort, 10 patients (25%) died during the follow up period. 8 patients required stem cell transplants, liver enzyme elevations were observed in these patients prior to the transplant. Causes for death included GI bleeding with hemorrhagic and septic shock in 1 patient, graft versus host disease post-transplant in 2, sepsis with multi-organ failure in 2. In the remaining 5 patients, causes of death remained unknown.
Discussion
In the largest cohort of patients with genetically confirmed telomere disease, we report a high prevalence of liver involvement. Approximately 40% of patients demonstrated hepatic manifestations either biochemically, radiologically, or histologically. In a small sampling of these patients by biopsy, pathologic diagnoses ranged from the more ubiquitous NAFLD to the rare case of nodular regenerative hyperplasia. Our cohort is also notable for PFT abnormalities. A majority of patients who had available PFTs had some degree of diffusion abnormality. This finding supports the association between short telomere disease and pulmonary abnormalities, specifically fibrosis.
An abnormal flow pattern was also noted in a significant number of patients with available PFTs. These data suggest that pulmonary abnormalities as detected by PFTs are prevalent among our cohort.
Several studies have discussed the role of telomere attrition in liver disease.(23–26) Kitada (25) and Urabe (26) described progressive reduction in telomere length with the progression of liver fibrosis. Aikata el al(27) showed that in younger patients with cirrhosis, there was marked excessive shortening in telomere length. Twenty nine (72.5%) subjects in our study had very short telomere lengths.
In contrast to pediatric phenotypes of dyskeratosis congenita in which bone marrow failure dominates clinical manifestations, telomere disease in adults manifests in organs with slow tissue-turnover, such as the lungs and liver. Defects in telomere repair may be foundational as the first of multiple ‘hits’ in addition to prolonged ongoing damage in long-lived cells that have limited ability to regenerate(28). We report a prevalence of up to 90% for lung diffusion abnormality and up to 52% for airflow impairment among patients with liver manifestations who had available PFTs. Our finding corroborates a similar finding by George et al (29) who reported abnormal liver biopsies (including nodular regenerative hyperplasia) in as well as imaging evidence of steatosis in patients with idiopathic pulmonary fibrosis and short telomeres.
Several studies have reported the presence of nodular regenerative hyperplasia in patients with telomere disease. Gorgy et al (15) reported an increased prevalence of hepato-pulmonary syndrome and nodular regenerative hyperplasia in patients with short telomeres and progressive dyspnea. Similarly, Calado et al, in their family study, showed that telomerase mutations were associated with histopathologic findings such as cryptogenic cirrhosis, nodular regenerative hyperplasia as well as massive hepatic necrosis. Unlike the studies above, we did not observe a high prevalence of NRH, perhaps because of a strong association of NRH with telomere disease characterized by pulmonary fibrosis. Non-alcoholic fatty liver disease, that was present in 23% of patients in our cohort, is in concordance with the prevalence of NAFLD in the US population. It is notable that despite the young age at diagnosis (median 42 years), a significant number had advanced liver disease, including NASH cirrhosis. Similarly, on liver biopsy, 3/6 patients had either bridging fibrosis or cirrhosis. These data should incite further research on the role of telomere shortening in acceleration of underlying liver disease as well as progression to cirrhosis.
Spleen to height ratios, while useful in other liver diseases (30,31), did not correlate with severity of liver disease, this is likely due to splenomegaly being part of the hematologic abnormalities related to this syndrome. Further investigation is required to evaluate novel non-invasive biomarkers in liver disease.
There are several limitations to our study. As this is a retrospective cohort study of patients seen intermittently at the NIH Clinical Center, some liver enzyme flares might not have been recorded due to patients receiving medical care elsewhere as well. Similarly, there is incomplete information about outcomes. Patients also underwent procedures including stem cell transplantation and received medications that could have potentially affected their liver. While an effort has been made to delineate medication-related liver enzyme elevations, drug induced liver injury remains an important component of the natural history in these patients. Another limitation is the small number of liver biopsies, potentially resulting in underreporting of some liver diseases, particularly NRH. Liver disease may be present even with normal liver enzymes(32), and would potentially be associated with a lower platelet count. In our cohort, severe thrombocytopenia precluded safe liver biopsies in several patients.
The fact that only 52% of our cohort had PFTs may reflect a selection bias. As PFTs were obtained for clinical reasons, the prevalence of abnormalities may represent an overestimation of the actual prevalence.
Our study has several strengths. It is a longitudinal study of patients with telomere disease, with a median follow up of 2.4 years. We have been able to report liver enzyme elevations that have persisted for over 6 months, as well as have been able to track resolution in case of drug induced liver injury. We have also been able to report liver-related outcomes in some of our patients.
In conclusion, the liver is involved in patients with telomere disease at much higher rates than previously appreciated, and these patients also have significant morbidity and mortality. Our clinical findings suggest that routine liver evaluations should be undertaken in patients with telomerase disease. Additionally, liver biopsy also plays an important role in this cohort, due to the multiplicity of findings on histopathology and the relative lack of other non-invasive predictors in a liver disease with systemic complications. Our study suggests that pulmonary evaluation in the form of PFTs should be considered in patients with telomere disease and liver manifestation as we report relatively high rates of liver-lung co-involvement. Further studies in other cohorts are needed to improve our understanding and characterization of the progression of liver disease in these patients.
Table 3:
Characteristics of liver enzyme elevations
| Patients (n=40) | Value |
|---|---|
|
Pattern of liver enzyme elevations Cholestatic Hepatocellular Mixed |
19(49%) 8(21%) 12(30%) |
|
Association with liver enzyme elevation Drug induced Iron overload Cirrhosis Steatosis GVHD Unknown |
8(20.5%) 5(12.5%) 4(10%) 5(12.5%) 1(2.5%) 10(27%) |
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