Ancestral mutation in telomerase causes defects in repeat addition processivity and manifests as familial pulmonary fibrosis

Abstract

The telomerase reverse transcriptase synthesizes new telomeres onto chromosome ends by copying from a short template within its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a property known as repeat addition processivity. However, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the age-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase reverse transcriptase gene hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first repeat addition, hTERT V791I-V867M showed severe defects in telomere repeat addition processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat addition processivity is a critical determinant of telomere length and telomere-mediated disease.

Figure 1. Position and conservation of non-synonymous variants in hTERT shared by pulmonary fibrosis families 13 and 143 probands.

A,B. Chromatograms of single nucleotide variants predict non-synonymous amino acid substitutions. The first was a c.2371G→A transition in exon 7 (A), and the second was c.2599G→A transition in exon 10 (B). C. Panel shows conserved hTERT motifs shared with other TERTs. The non-synonymous amino acid variant residues are indicated within the reverse transcriptase domain. hTERT V791 falls in the IFD domain between the A and B motifs, and hTERT V867 is adjacent to the invariant motif C aspartic acid residues which are essential for reverse transcriptase function and are indicated by *. Alignment of TERT sequence across 14 species indicates that both V791 and V867 fall within conserved motifs.

Figure 2. Four generation pedigrees of pulmonary fibrosis probands from Families 13 and 143 of the Vanderbilt Registry.

Mutation and affected status are indicated by symbols shown in the key and individuals in whom DNA was available are noted by the pedigree number in bold text. In both families, the hTERT 791I and 867 M variants co-segregate, consistent with the fact that these heterozygous substitutions are on the same allele in cis. hTERT 791I-867M also segregates with the pulmonary fibrosis phenotype in all the individuals in whom DNA was available. The symbols are identified in the key, and completely filled symbols indicate clinically affected individuals who carry the double mutant TERT.

Figure 3. The ancestral mutant telomerase affects repeat addition processivity in vitro.

A. Telomerase activity assay of non-synonymous hTERT variants identified in family 13 and 143 probands. L55Q was previously identified in a pulmonary fibrosis family and known to compromise catalysis. Telomerase activity assay at high nucleotide (1 mM) concentrations on the left shows no defects in catalytic activity or processivity for hTERT V791I and V867M or the double mutant. At lower nucleotide concentration (10 µM), hTERT 867M and hTERT V791I-V867M both show defects in repeat addition processivity as evidenced by the decreased intensity of the high molecular weight products relative to the first repeat. Low exposure image of the internal loading control is shown below. B. Low exposure image of the gel shown in (A) is shown to visualize the +1, +2, and +3 nucleotide bands clearly. C. SDS-PAGE of ³⁵S labeled hTERT used in (A) to monitor the expression of in vitro synthesized hTERTs. D. Quantitation of first repeat addition as measured by the total intensity of the +1, +2, +3, and +4 nucleotide bands. Quantitation is based on 3 independent experiments. * Indicates P-value <0.01 and error bars indicate standard error of the mean. E. Quantitation of processivity across the first four repeats (R₁, R₂, R₃ and R₄) is shown by the linear regression line.

Figure 4. Telomere length in mutation carriers in families 13 and 143 have short telomeres compared to age-matched controls.

Panel shows telomere length as measured in lymphocytes by flow-FISH compared to normal distribution of age-matched controls. Percentiles are based on telomere length data from 400 controls. Squares refer to males and circles refer to females. Individuals refer to pedigree position in Figure 2. Individual 13III.8 has short telomeres and was diagnosed with an overlap syndrome of emphysema and pulmonary fibrosis.

Figure 5. CAT scans from pulmonary fibrosis probands and non-carrier sibling with short telomeres.

A,B show lower thoracic CAT scan images from the probands in family 13 and 143. Both images show basilar honeycombing typical of idiopathic pulmonary fibrosis. C,D are apical and lower thoracic CAT scan images respectively of sibling with short telomere who does not carry the mutant hTERT (Individual designated 13III.8 in Figure 2 and Figure 3). This individual has apical changes consistent with centrilobular emphysema as well as lower thoracic ground glass changes consistent with an interstitial process.