Telomere Biology Gene Mutation and Transplant Outcome in Patients with Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome known as the prototype of telomere diseases. In addition to the clinical triad (nail dystrophy, hyperpigmentation, and leukoplakia), very short telomeres (below the 1^st percentile) is a marker for the diagnosis of DC (Calado & Young, NEJM 2009). Telomere dysfunction was associated with DC after discovery of DKC1 mutations in patients presenting the X-linked form of the syndrome. Novel mutations in telomere biology genes (TERC, TERT, NOP10, NHP2, TINF2, TCAB1, CTC1, RTEL1, ACD, and PARN) have been described in patients with DC. Variations in all these genes can affect telomere protection and maintenance, leading to telomere shortening and development of telomeropathies. In this study, we mapped the TERT, TERC, DKC1, and TINF2 genes for mutations in 15 patients (median age = 10 years; M/F = 11/4) with DC and very short telomeres, in order to classify these cases in a molecular level and determine the frequency of these mutations in our cohort. Survival for twelve patients who underwent allogeneic hematopoietic stem cell transplant (HSCT) was assessed and correlated with mutational status and telomere length. Diagnosis of DC was made according to the definition of Calado & Young (2009). Telomere length was measured in nucleated blood cells by flow-FISH and mutational screening was performed on genomic DNA extracted from peripheral blood cells by direct sequencing. Seven non-synonymous mutations were identified in TINF2 (40%), two in DKC1 (13%), one in TERT (6%), and one in TERC (6%). The TINF2 variants R282H and R282C had been already described as pathogenic, as well T66A and A353V DKC1 variants (Knight et al, 1999; Savage et al, 2008; Walne et al, 2008). The heterozygous variant R282H (c. 845 G>A) in TINF2 were found in 4 unrelated patients. One of them also harbor the variants Q120R and Q157H in the same gene. The heterozygous mutation in TINF2 R282C (c. 844 C>T) was found in one patient, that also presented the common polymorphism A279T in TERT. The pathogenic variants T66A (c.196A>G) and A353V (c.1058C>T) in DKC1 were found in two different male patients. Moreover, three novel mutations were identified in our cohort, r.94 C>T in TERC, F290C in TINF2, and R696Cin TERT. The heterozygous mutation r.94 (C>T) found in TERC was located at the pseudoknot P2b region of the gene and the patient who carries that presented a severe aplastic anemia and all DC clinical triad. The novel heterozygous F290C (c.859 T>G) variant is located at the "hot spot" in exon 6 of TINF2 andwas found in one patient that presented a severe phenotype of DC. In silico analysis with SIFT and Polyphen-2 predicted that this variant is not tolerated and probably damaging, which is consistent with the pathogenicity of the mutation. The homozygous mutation R696C (c.2086 C>T) in TERT was found in one patient and also in his two brothers. All of them presented reduced blood cell count, clinical features of DC, and severe aplastic anemia. The family screening identified the father and sister as heterozygous for the same mutation, but both asymptomatic. DNA sample from the mother were not available for this study. In silico analysis by SIFT and Polyphen 2.0, predicted that the R696C mutation is not tolerant and possible damaging to telomerase activity, respectively. To validate in silico analysis, TRAP assay with cell lysates obtained from telomerase-negative VA13 cell line transfected with wild type or R696C mutated TERT vector and TERC vector is under evaluation. Consistently with previously studies, telomere length in patients with TINF2 mutations were the shortest compared with the other telomeres genes mapped in this study. Although, the phenotype and severity of the disease does not appear to change according to the mutated gene. Also, the mutational status (p=0.28) or telomere length (p=0.21) did not influence the survival rates of patients after HSCT.

Flow-FISH was able to identify patients with very short telomeres and validated telomere length measurement as a diagnostic tool for DC. Direct sequencing of the most commonly mutated genes in DC in a cohort of patients with telomeres below 1^st percentile was able to characterize the genetic cause of this disease in more than 70% of the cases. The identification of genetic defect in DC can manage clinical decisions and is essential to genetic counseling prior to bone marrow transplantation.