TINF2 Mutations In Patients with Aplastic Anemia Result In Low TIN2 Expression In Hematopoietic Cells and Very Short Telomeres.

Abstract 1165 Telomeres, the ends of chromosomes, protect against chromosomal instability. They are composed of hundreds to thousands TTAGGG double-stranded DNA repeats coated by at least six telomere-binding proteins collectively called “shelterin” (TRF1, TRF2, TIN2, TPP1, and POT1). As telomeres shorten at each cell division, highly proliferative cells express telomerase, a reverse transcriptase that elongates telomere ends. Loss-of-function mutations in genes encoding proteins and RNA of the telomerase complex are etiologic in dyskeratosis congenita (DC) and also are genetic risk factors for acquired aplastic anemia (AA), cancer, familial idiopathic pulmonary fibrosis, and liver diseases (cirrhosis and nodular regenerative hyperplasia). More recently, mutations in the shelterin component TIN2 were reported in patients with DC, further supporting that short and dysfunctional telomeres are the molecular etiology of marrow failure in dyskeratosis congenita. In order to investigate whether TINF2 mutations occurred in individuals with apparently acquired AA, we screened 190 consecutive patients diagnosed with the disease based on the International Agranulocytosis and Aplastic Anemia Study, all of whom were treated at a single institution (Hematology Branch, National Institutes of Health). All six TINF2 exons were bi-directionally sequenced using the Big Dye® sequencing method. We have observed eleven synonymous polymorphisms in TINF2 exons. Additionally four nonsynonymous TINF2 gene variants were identified in seven patients (carrier frequency, 3.7%). Three patients carried both codon Gly237Asp and codon Pro241Ser polymorphisms (one homozygous and two heterozygous); two patients carried codon Gly237Asp alone; one patient was heterozygous for a novel codon Gln120Arg TINF2 mutation; and one patient was heterozygous for a novel Thr275fs*codon316X TINF2 mutation. Patients carrying TINF2 nonsynonymous variants were significantly younger (median age, 13 years; range, 4–23 years vs. 36 years; 2–78 years; p=0.0015) but none had any signs of classic DC. Peripheral blood lymphocyte9s telomere length measured by flow-fluorescence in situ hybridization was extremely short in the patient with a novel frameshift mutation (2.7 kb; expected-for-age length, 9.2 kb; below 1st percentile) and short in the patient with the novel codon Gln120Arg mutation (6.6 kb; expected-for-age, 8.0 kb; below the 10th percentile). Telomeres were short (below the 10th percentile) in three out of five patients carrying codon Gly237Asp and/or codon Pro241Ser variants. Hematopoietic cells of healthy volunteers and patients with TINF2 gene variants were probed for TIN2 expression in bone marrow samples by immunohistochemistry (polyclonal anti-TIN2 kindly provided by Dr. Titia de Lange, Rockefeller University). In normal bone marrows, TIN2 stained positive in megakaryocytes and in myeloid progenitors in a nuclear pattern. Mature granulocytes and lymphocytes were negative for TIN2 expression. Erythroid progenitors stained negative in the nucleus but some displayed a blushed positivity in the cytoplasm, which was demonstrated to be non-specific in immunoblot analyses of nuclear and cytoplasmic cell lysates. In the two patients with novel mutations, hematopoietic cells stained negative for TIN2. In patients carrying the two variants Gly237Asp and Pro241Ser, TIN2 stained weak or negative in hematopoietic cells, although it appeared in endothelial cells in the bone marrow. Patients with DKC1 or TERT mutations also were analyzed as controls and stained positive in the nucleus, similar to healthy controls, in spite of having very short telomeres. Our results describe for the first time the pattern of TIN2 expression in hematopoietic cells. TIN2 is positive with a nuclear pattern in megakaryocytes and early myeloid precursors, but not in erythroid progenitors or lymphocytes. The present findings further supports the relevance of the shelterin complex for telomere stability and for disease susceptibility; TINF2 mutations which result in short telomeres are present at relatively low frequencies in patients with apparently acquired AA but have important clinical management implications. TINF2 mutations are associated with reduced TIN2 expression in hematopoietic progenitors in the marrow. Disclosures: Lansdorp: Repeat Diagnostics: Equity Ownership.