Constitutional telomerase mutations are genetic risk factors for cirrhosis

Abstract

Some patients with liver disease progress to cirrhosis, but the risk factors for cirrhosis development are unknown. Dyskeratosis congenita, an inherited bone marrow failure syndrome associated with mucocutaneous anomalies, pulmonary fibrosis, and cirrhosis, is caused by germline mutations of genes in the telomerase complex. We examined whether telomerase mutations also occurred in sporadic cirrhosis. In all, 134 patients with cirrhosis of common etiologies treated at the Liver Research Institute, University of Arizona, between May 2008 and July 2009, and 528 healthy subjects were screened for variation in the TERT and TERC genes by direct sequencing; an additional 1,472 controls were examined for the most common genetic variation observed in patients. Telomere length of leukocytes was measured by quantitative polymerase chain reaction. Functional effects of genetic changes were assessed by transfection of mutation-containing vectors into telomerase-deficient cell lines, and telomerase activity was measured in cell lysates. Nine of the 134 patients with cirrhosis (7%) carried a missense variant in TERT, resulting in a cumulative carrier frequency significantly higher than in controls (P = 0.0009). One patient was homozygous and eight were heterozygous. The allele frequency for the most common missense TERT variant was significantly higher in patients with cirrhosis (2.6%) than in 2,000 controls (0.7%; P = 0.0011). One additional patient carried a TERC mutation. The mean telomere length of leukocytes in patients with cirrhosis, including six mutant cases, was shorter than in age-matched controls (P = 0.0004).

Conclusion: Most TERT gene variants reduced telomerase enzymatic activity in vitro. Loss-of-function telomerase gene variants associated with short telomeres are risk factors for sporadic cirrhosis.

Figure 1

(A) Schematic domain structure of TERT, indicating 3 major regions: N-terminal, reverse transcriptase motifs, and C-terminal. Mutation codon location and amino acid substitutions caused by mutations are shown. Abbreviations for aminoacid residues: A, alanine; E, glutamic acid; H, I, isoleucine; L, leucine; P, phenylalanine; T, threonine. (B) Leukocyte telomere length was measured by quantitative polymerase chain reaction (qPCR). Telomere length of 175healthy controls is shown in gray and patients with telomerase mutation and hepatic cirrhosisare depicted indifferent colors. (C) Telomere length of cirrhotic patients without identifiable mutations (red) was significantly shorter than in healthy controls (gray; P=0.0004). Eighty-two percent of cirrhotic patients had their telomere lengths below the median. (D)Telomerase activity of the empty (Flag), wild-type, or mutated TERT or TERC expression vectors in the telomerase-negative VA13 cell line was measured by the fluorescent telomeric repeat-amplification protocol (TRAP) assay. Telomerase activity was considered 100% for the wild-type. Telomerase activity in each experiment was corrected for TERT or TERC mRNA levels as measured by RT-PCR.