Familial Clonal Hematopoiesis in a Long Telomere Syndrome

Abstract

Background: Telomere shortening is a well-characterized cellular aging mechanism, and short telomere syndromes cause age-related disease. However, whether long telomere length is advantageous is poorly understood.

Methods: We examined the clinical and molecular features of aging and cancer in persons carrying heterozygous loss-of-function mutations in the telomere-related gene POT1 and noncarrier relatives.

Results: A total of 17 POT1 mutation carriers and 21 noncarrier relatives were initially included in the study, and a validation cohort of 6 additional mutation carriers was subsequently recruited. A majority of the POT1 mutation carriers with telomere length evaluated (9 of 13) had long telomeres (>99th percentile). POT1 mutation carriers had a range of benign and malignant neoplasms involving epithelial, mesenchymal, and neuronal tissues in addition to B- and T-cell lymphoma and myeloid cancers. Five of 18 POT1 mutation carriers (28%) had T-cell clonality, and 8 of 12 (67%) had clonal hematopoiesis of indeterminate potential. A predisposition to clonal hematopoiesis had an autosomal dominant pattern of inheritance, as well as penetrance that increased with age; somatic DNMT3A and JAK2 hotspot mutations were common. These and other somatic driver mutations probably arose in the first decades of life, and their lineages secondarily accumulated a higher mutation burden characterized by a clocklike signature. Successive generations showed genetic anticipation (i.e., an increasingly early onset of disease). In contrast to noncarrier relatives, who had the typical telomere shortening with age, POT1 mutation carriers maintained telomere length over the course of 2 years.

Conclusions: POT1 mutations associated with long telomere length conferred a predisposition to a familial clonal hematopoiesis syndrome that was associated with a range of benign and malignant solid neoplasms. The risk of these phenotypes was mediated by extended cellular longevity and by the capacity to maintain telomeres over time. (Funded by the National Institutes of Health and others.).

Figure 1 (facing page).. Telomere Length and Pedigrees of POT1 Mutation Carriers.

Panel A shows telomere lengths measured by flow cytometry and fluorescence in situ hybridization in 13 living POT1 mutation carriers and their noncarrier relatives. Data are plotted relative to a clinically validated nomogram derived from healthy controls. Some data points differ in size in order to make overlapping data points visible. Panel B shows pedigrees of POT1 mutation carriers. Probands are indicated with arrows. Bold identifiers indicate persons for whom data on the POT1 genotype were available. Circles denote female family members, and squares male family members; a line through a symbols indicates that the person is deceased. Panel C shows POT1 with mutations annotated relative to conserved domains. HJRL denotes holiday junction resolvase-like, and OB oligonucleotide-binding. Panel D shows an immunoblot of endogenous POT1 levels in lymphoblastoid cell lines with levels quantified below in arbitrary units relative to tubulin (replicate data in Fig. S1A). Panel E shows an electrophoretic mobility-shift assay (EMSA) for POT1 missense mutations. The ΔOB mutant is truncated for amino acids 127 through 635. POT1R273L, a variant reported in familial melanoma, was included as a positive control. DNA binding is quantified below as a proportion relative to wild type, and results were replicated twice. IVT denotes in vitro translated, and oligo oligonucleotide.

Figure 2.. Benign and Malignant Neoplastic Manifestations among POT1 Mutation Carriers.

Panel A shows the diagnoses among 17 POT1 mutation carriers and the ages at last assessment. Age at death is indicated by “d.” The clonal hematopoiesis in all persons shown here met the threshold for clonal hematopoiesis of indeterminate potential (CHIP; i.e., variant-allele frequency of ≥2%); for four deceased persons and one person who was recruited at the end of the study, clonal hematopoiesis and T-cell clonality were not assessed. M denotes male and F female. Panel B shows a representative image of benign melanosis (arrow). Panel C shows a computed tomography (CT)–captured goiter (arrow). Panel D shows an axial CT image of a left kidney mass diagnosed as clear-cell carcinoma (arrow). Panel E shows a representative magnetic resonance image of pediatric-onset glioblastoma multiforme. Panel F shows the range of benign to malignant diagnoses grouped according to the affected tissue among 17 POT1 mutation carriers, with the number of affected persons in Panel A shown in parentheses.

Figure 3 (facing page).. Lymphoid and Myeloid Clonality among POT1 Mutation Carriers.

Panel A shows images (hematoxylin and eosin staining) of small bowel from an autopsy examination of a POT1 mutation carrier in whom large T-cell lymphoma developed after receipt of immune-checkpoint inhibitor therapy for metastatic melanoma. Lymphocyte infiltrates involving the lamina propria were positive for CD3. Panel B shows productive clonality scores plotted against age (calculated from T-cell receptor Vβ CDR3 sequencing as 1 minus the normalized Shannon’s entropy for all productive rearrangements). Scores range from 0 to 1, where 0 represents polyclonality and 1 indicates one rearrangement dominating the entire repertoire. The 18 POT1 mutation carriers are from seven unrelated families. Percentile lines are derived from 258 healthy bone marrow donors (controls) who were seropositive for cytomegalovirus, as derived from Emerson et al. One person with a history of cutaneous T-cell lymphoma but no known systemic disease is indicated with a dagger. Panel C shows a CHIP co-mutation plot including POT1 mutation carriers and their noncarrier relatives sorted according to increasing age with the germline mutations indicated below. JAK2 GGCC haplotype status is also color-coded in the key. The three persons indicated with an asterisk had additional DNMT3A mutations with variant-allele frequencies of 1% or higher, one of whom had two DNMT3A mutations with variant-allele frequencies of 2% or higher; all these mutations are shown in Panel D. Panel D shows DNMT3A mutations identified in POT1 mutation carriers and in one related noncarrier relative to the conserved domains of the protein and their allele frequency. Filled circles denote nonsynonymous mutations and the unfilled circle a splicing variant. Panel E shows JAK2 V617F variant-allele frequencies identified by targeted ultradeep sequencing in POT1 mutation carriers and their noncarrier relatives, shown in relation to lymphocyte telomere length.

Figure 4.. Phylogenies of Hematopoietic Colonies.

Panels A, B, and C show phylogenies of single cell–derived hematopoietic colonies on a somatic variant scale (single-nucleotide variants and short insertions–deletions). The POT1 genotype, the person’s age, and the telomere length (TL) percentile are shown above each tree. In Panel B, two de novo JAK2 mutations are resolved in the tree, and the two cooccurring DNMT3A mutations in Panel C were found to be biallelic in phasing studies. The tree topology in the two POT1 mutation carriers shows the tips, which represent contemporary hematopoietic colonies that are descended from fewer ancestral progenitor lineages (internal branches), in contrast to the tree of the related noncarrier shown in Panel A. The number of somatic variants in POT1 mutation carriers was also higher than in the noncarrier, even in clades without detectable driver mutations.

Figure 5 (facing page).. Genetic Anticipation and Longitudinal Change in Telomere Length among POT1 Mutation Carriers.

Panel A shows the age at assessment and cancer diagnosis in three pedigrees. Mutation carriers (shaded) carry POT1 p.R273Q in Pedigrees 1 and 3 and carry p.I78T in Pedigree 2. One mutation carrier in Pedigree 1 had three separate melanoma diagnoses. Panels B and C show differences in lymphocyte and granulocyte telomere lengths relative to the median for age at study enrollment and at 2 years. Data are shown for POT1 mutation carriers and their siblings and children who are noncarriers. Panel D shows the mean absolute change in telomere length over a 2-year period in POT1 mutation carriers (red) and their noncarrier relatives (blue). Error bars indicate the standard error. Panel E shows the mean variant-allele frequency for a somatic heterozygous driver mutation acquired at birth over a lifetime in telomere length groups calculated from 10,000 simulations for each group. The dashed line indicates the 2% variant-allele frequency threshold defining CHIP. Panel F shows contrasting hematopoietic and immune phenotypes associated with POT1 mutations (long telomere length) and short telomere syndromes. Each dot represents a hypothetical person with a germline telomere maintenance defect plotted relative to the normal telomere length distribution in the human population. This panel was adapted from Armanios.