Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita

Abstract

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which the known susceptibility genes (DKC1, TERC, and TERT) belong to the telomere maintenance pathway; patients with DC have very short telomeres. We used multicolor flow fluorescence in situ hybridization analysis of median telomere length in total blood leukocytes, granulocytes, lymphocytes, and several lymphocyte subsets to confirm the diagnosis of DC, distinguish patients with DC from unaffected family members, identify clinically silent DC carriers, and discriminate between patients with DC and those with other bone marrow failure disorders. We defined "very short" telomeres as below the first percentile measured among 400 healthy control subjects over the entire age range. Diagnostic sensitivity and specificity of very short telomeres for DC were more than 90% for total lymphocytes, CD45RA+/CD20- naive T cells, and CD20+ B cells. Granulocyte and total leukocyte assays were not specific; CD45RA- memory T cells and CD57+ NK/NKT were not sensitive. We observed very short telomeres in a clinically normal family member who subsequently developed DC. We propose adding leukocyte subset flow fluorescence in situ hybridization telomere length measurement to the evaluation of patients and families suspected to have DC, because the correct diagnosis will substantially affect patient management.

Figure 1

Telomere length according to age in patients with dyskeratosis congenita and their relatives. The vertical axis represents telomere length in kilobytes. Lines in the figures indicate the first, tenth, 50th, 90th, and 99th percentiles of results from 400 normal control subjects. Symbols represent subjects: 17 patients with dyskeratosis congenita (red solid circle), 4 Hoyeraal-Hreidarsson variant (green solid triangle), 4 Revesz syndrome (turquoise solid diamond), one silent carrier (blue solid square), and 54 relatives (open square). (Top panels) Granulocytes, lymphocytes, and CD45RA-positive/CD20-negative naive T cells. (Bottom panels) CD45RA-negative memory T cells, CD20-positive B cells, and total leukocytes.

Figure 2

Telomere length according to age in non–dyskeratosis congenita patients and their relatives. The vertical axis represents telomere length in kilobytes. Lines in the figures indicate the first, tenth, 50th, 90th, and 99th percentiles of results from 400 normal control subjects. Symbols represent subjects: 13 Fanconi anemia (FA) patients (red solid circle), one FA postbone marrow transplant (blue solid circle), 3 FA mosaics (turquoise solid circle), 14 Diamond-Blackfan anemia (green solid triangle), 5 Shwachman-Diamond syndrome (black solid diamond), 10 other (magenta solid square), and 35 relatives (open square).

Figure 3

Telomere length according to age in dyskeratosis congenita and non–dyskeratosis congenita patients. The vertical axis represents telomere length in kilobytes. Lines in the figures indicate the first, tenth, 50th, 90th, and 99th percentiles of results from 400 normal control subjects. Symbols represent subjects: 26 patients with dyskeratosis congenita (red solid circle), 46 non–dyskeratosis congenita patients (black solid triangle).

Figure 4

Regression lines for telomere length according to age. The vertical axis represents the median telomere length in kilobytes. Lines in the figures indicate the first, tenth, 50th, 90th, and 99th percentiles of results from 400 normal control subjects. Patients with dyskeratosis congenita (DC) (red solid line), relatives with dyskeratosis congenita (red dashed line), non–dyskeratosis congenita patients (black long dashed line), and non–dyskeratosis congenita relatives (black short dashed line).