Secondary Logo

Journal Logo


Fanconi anemia

studying telomeres and telomerase genes using fluorescent in-situ hybridization technique in Egyptian patients

Temtamy, Samia A.a; Eid, Maha M.b; Mohamed, Amal M.b; Kayed, Hesham F.b; Shihab, Marwa I.b; El-Kamah, Ghadaa

Author Information
doi: 10.1097/01.MJX.0000397209.85926.70
  • Free



Fanconi anemia (FA) is an autosomal recessive and X-linked disease that is characterized by multiple congenital abnormalities, bone marrow failure, and a high risk of malignancies [1]. Somatic cell fusion studies show that FA is genetically heterogeneous, with at least 13 complementation groups identified thus far [2]. The most important clinical features of FA are hematological. Children with FA often develop pancytopenia during the first few years of life. Complications of bone marrow failure are the major causes of morbidity and mortality of FA, and 80% of patients with FA die from bone marrow failure [3].

Patients with FA display progressive reduction in cell growth capacity, increased genomic instability, and high levels of apoptosis [4]. Consistent with this, an apparent accelerated shortening of telomeres has been reported in patients with FA and in other hematopoietic disorders, such as acquired aplastic anemia (AA) and dyskeratosis congenital [5].

Telomeres play important roles in genome stability. Telomere integrity is also crucial for organism viability and in particular for normal functioning of the hematopoietic system [6]. Telomere length and telomerase activities have been implicated in the control of cell proliferation [7]. Telomere shortening represents both replicative and environmental (oxidative and other) stresses. Telomere shortening during cell division can be attenuated by the activity of telomerase, a reverse transcriptase that adds TTAGGG repeats to telomeres on DNA synthesis. As ageing of cells is associated with shortening of telomeres and is accelerated by reactive oxygen species, it was assumed that if FA is a disorder of reactive oxygen species metabolism, telomere shortening should be accelerated in patients with FA. Indeed, accelerated telomere shortening was found in primary cultured FA fibroblasts and was attributed to oxidative stress [8].

Telomerase activation was known to be present in FA, as in other pathologies in which telomeres are shortened. Different theories have been postulated to explain telomerase reactivation in patients with FA. An increase in telomerase activity has been reported during cell proliferation [7] and also may be enhanced during the first step in hemopoietic cell differentiation [9]. Thus, telomerase induction in FA cells could possibly be due to free radicals and might be a consequence of hyperstimulated hemopoiesis. Reduction of cell proliferation in response to telomere shortening would be expected to be progressive. When the ability of hemopoietic stem/progenitor FA cells to proliferate becomes too impaired due to telomere shortening, progressive pancytopenia occurs. In this second step, telomere shortening would play a direct role in the evolution toward pancytopenia [8].

In light of the importance of the telomere/telomerase system in genomic stability and its relevance to the development of pancytopenia, it was found necessary to evaluate the telomeric ends in patients with FA and the telomerase genes (TERC and TERT genes) as a potential cause for the telomerase enzyme reactivation in patients with FA.



Blood samples were obtained from seven patients with FA (four female and three male), three patients with acquired AA (two male and one female) and five healthy individuals in the control group. Informed consents were taken from the parents of children included in the study according to the guidelines of the ethics committees of the National Research Centre, Egypt. The patients were subjected to thorough clinical evaluation, pedigree analysis, anthropometric measurements that included height, weight, and head circumferences and complete blood count.

Cytogenetic studies

Blood culture

Whole blood was cultured in four 9 ml culture tubes with complete culture media and was incubated for 72 h.

Diepoxybutane test

DEB was added to only two culture tubes 48 h before harvesting. DEB is essential for the diagnosis of FA.

Fluorescence in-situ hybridization analysis

All human telomere probes: In-situ hybridization of metaphase chromosomes was carried out on one slide using all human telomere (spectrum orange) fluorescent in-situ hybridization (FISH) probes (Q Biogene, USA) specific for the human telomere repeat sequence (TTAGGG) according to the manufacture's instructions. The captured metaphases were used to count the telomeric ends without signals, and extratelomeric signals (intrachromosomal and extrachromosomal).

TERC and TERT gene FISH probes: hTERC (3q26/cMYC 8q24) and hTERT (5p15/5q31) gene FISH probes (Kreatech, Amsterdam, The Netherlands) were applied on two separate slides for each case to investigate the telomerase genes in FA and non-FA patients.

Slides were examined by using an applied imaging system, that is, an Olympus BX51 microscope (Olympus, Tokyo, Japan) with a fluorescent attachment and equipped with filter sets.

Statistical analyses

Mann–Whitney test was used to compare between the patients with FA and the control group with respect to telomeric deletion and extratelomeric signals. In addition, the Pearson test was used for correlation studies.


The clinical findings of the patients are summarized in Table 1. The hemoglobin level for the patients ranged from 5.5 g to 8.1 g/dl, the RBC count ranged form 1 600 000 to 2 300 000/cmm, and the WBC count for all the patients was within the normal value. The most striking was the platelet count; their values were much below the normal (10 000–40 000/cmm).

Table 1:
Summary of the clinical feature of the patients

According to the DEB test, the patients were classified into seven DEB positive (FA) and three DEB negative (non-FA).

The results of FISH technique using all human telomeres probe for the patients with FA, non-FA, and the control groups are summarized in Tables 2–4.

Table 2:
Results of FISH technique using AHT probe for patients with FA
Table 3:
Results of FISH technique using AHT probe for non-FA patients
Table 4:
Results of FISH technique using AHT probe for the normal control group

At first, the telomeric end deletions were counted for the FA, non-FA, and the control groups. High frequencies of telomeric ends without signals were detected in FA and non-FA groups (Figs 1–3). The mean values of the telomeric deletion for the FA, non-FA, and the control groups were 23.1, 16.6, and 6.4, respectively. The statistical analysis of the data using Mann–Whitney test revealed no significant difference between FA and non-FA (P=0.108). In contrast, a highly significant difference was found between FA and the control groups (P=0.003), and a slightly significant difference between non-FA and the normal groups (P=0.05). Then the extra telomeric signals were counted (intrachromosomal and extrachromosomal). The mean values for the intrachromosomal signals for FA, non-FA, and control were 4.8, 5, and 2.2 respectively, and the mean values of the extrachromosomal signals in the same order were 2.9, 3.8, and 0.44, respectively. In addition, there was no significant difference between FA and non-FA regarding extrachromosomal and intrachromosomal signals (P=0.5 and 0.9 respectively), but there was significant difference between FA and control and non-FA and the control groups regarding the extrachromosomal signals (P=0.003 and 0.01, respectively).

Fig. 1:
Fluorescent in-situ hybridization using all human telomere probe show telomeric ends without signals in a patient with Fanconi anemia.
Fig. 2:
Fluorescent in-situ hybridization using all human telomere probe show telomeric ends without signals in a patient with non-Fanconi anemia.
Fig. 3:
Fluorescent in-situ hybridization using all human telomere probe show telomeric ends without signals and increase intrachromosomal signals in patients with Fanconi anemia.

By applying the Pearson correlation coefficient test to find out if there is any correlation between the clinical findings of the patients and the telomeric deletion, an inverse correlation was found between the telomeric deletion and the age of onset of the anemia (r=−0.13) as well as the platelets and hemoglobin levels (r=−0.8 and −0.9, respectively). In contrast, there was direct relationship between telomeric deletion and the number of the congenital anomalies present in the patient (r=0.2).

The results of both TERC and TERT genes were negative; there was no gene amplification in either genes.


Telomeres, repeated sequences at the ends of chromosomes, are protective chromosomal structures highly conserved from primitive organisms to humans. Telomeres inevitably shorten with every cell cycle, and telomere attrition has been hypothesized to be fundamental to normal senescence of cells, tissues, and organisms [10]. Although FA and AA have different etiological factors, in our patients the telomeric end deletions were detected in a significant value for both groups, without any significant difference between them. As it was reported in the previous studies in the literature, compared with controls, accelerated shortening of telomere repeats or enhanced loss of telomere signals of FA lymphocytes and FA fibroblasts were detected [5,11–13]. Different theories have been postulated to clarify the underlying causes for the deletions [13]. As FA is one of the chromosomal breakage syndromes, breakage at the telomeric ends was suggested to be one of the contributing factors in the telomeric deletion in FA and AA. This theory was supported by the presence of increased extratelomeric signals (intrachromosomal and extrachromosomal) in our patients. However, because the number of deletions exceeded the number of extratelomeric signals, the accelerated replicative telomeric erosion was also suggested [13].

In contrast, a direct relationship has been postulated between telomeric deletion, telomerase overexpression, and oxidative stress [2,4]. Thus, oxidative stress could be one of the factors that led to telomeric erosion in patients with FA. In our study, telomeric deletion may reflect the severity of the condition of the patients. This finding is based on the presence of reverse correlation between the age of onset of anemia and telomeric deletion, which means that the earlier the age at onset of the anemia the more increase in the telomeric deletion. To our knowledge, this finding was not reported before. In addition, an inverse correlation was detected between telomeric deletion and the severity of anemia. This finding is perfectly matched to a previous study reported in the literature [11].

We can conclude that the increase in the level of telomeric deletion is associated with the decrease in the age of developing anemia and decrease in the levels of both platelets and hemoglobin and increased number of congenital anomalies.

It is worth mentioning that the patient with the highest level of deletion with average 28 telomeric end deletion had the earliest age at onset of anemia (1 year) and the most decreased levels of hemoglobin 5.5 g/dl and platelets 10 000/cmm and the highest number of congenital abnormalities affecting most of her systems (eight congenital abnormalities).

Several studies have detected an increase in the telomerase activity in FA. This was suggested to be due to a response to the telomeric deletion [2,4], because the reconstitution of telomerase activity may protect telomeres from further erosion [2,4]. In contrast, some studies have detected mutation in the telomerase genes [14,15]. Therefore, we thought it is essential to evaluate the hTERT and hTERC genes in the patients with the telomeric shortening, to investigate whether the increase in the telomerase activity previously reported in patients with FA is associated with telomerase genes amplification or not [4].

In this study, none of our patients exhibited any increase in the telomerase gene copy number. This finding confirms that the reactivation in the telomerase enzyme previously reported in FA is not associated with copy number increase in the telomerase genes. In contrast to malignancies, the increased level of telomerase enzymes is usually associated with amplification of the telomerase genes either one or both genes [16].

From our results, we can conclude that telomeric deletion could be used as a prognostic marker for patients with FA and AA. Examination of the telomeric ends may give an idea about the progress of the disease. In patients with intact telomeres, we can predict a better course of the disease and vice versa. Thus, because of the importance of this finding we recommend the use of the telomeric probe to be applied on a wider scale as a prognostic marker for patients with FA and AA. In contrast, because oxidative stress could be one of the factors that led to telomeric deletion [17], treatment with antioxidant drugs could be beneficial for such patients by reducing the accelerating rate of telomeric shortening.


1. Li X, Leteurtre F, Rocha V, Guardiola P, Berger R, Daniel MT, et al. Abnormal telomere metabolism in Fanconi's anaemia correlates with genomic instability and the probability of developing severe aplastic anaemia. Br J Haematol. 2003;120:836–845
2. Du W, Adam Z, Rani R, Zhang X, Pang Q. Oxidative stress in Fanconi anemia hematopoiesis and disease progression. Antioxid Redox Signal. 2008;10:1909–1921
3. Bagby GC Jr. Genetic basis of Fanconi anemia. Curr Opin Hematol. 2003;10:68–76
4. Leteurtre F, Li X, Guardiola P, Le Roux G, Sergère JC, Richard P, et al. Accelerated telomere shortening and telomerase activation in Fanconi's anaemia. Br J Haematol. 1999;105:883–893
5. Hanson H, Mathew CG, Docherty Z, Mackie Ogilvie C. Telomere shortening in Fanconi anaemia demonstrated by a direct FISH approach. Cytogenet Cell Genet. 2001;93:203–206
6. Lee HW, Blasco MA, Gottlieb GJ, Horner JW, Greider CW, DePinho RA. Essential role of mouse telomerase in highly proliferative organs. Nature. 1998;392:569–574 II
7. Greider CW. Telomere length regulation. Annu Rev Biochem. 1996;65:337–365
8. Uziel O, Reshef H, Ravid A, Fabian I, Halperin D, Ram R, et al. Oxidative stress causes telomere damage in Fanconi anaemia cells: a possible predisposition for malignant transformation. Br J Haematol. 2008;142:82–93
9. Norrback KF, Roos G. Telomeres and telomerase in normal and malignanthaematopoietic cells. Eur J Cancer. 1997;33:774–780
10. Calado RT. Telomeres and marrow failure. Hematology Am Soc Hematol Educ Program. 2009;1:338–343
11. Ball SE, Gibson FM, Rizzo S, Tooze JA, Marsh JC, Gordon-Smith EC. Progressive telomere shortening in aplastic anemia. Blood. 1998;91:3582–3592
12. Adelfalk C, Lorenz M, Serra V, Von Zglinicki T, Hirsch-Kauffmann M, Schweiger M. Accelerated telomere shortening in Fanconi anemia fibroblasts: a longitudinal study. FEBS Lett. 2001;506:22–26
13. Callén E, Samper E, Ramírez MJ, Creus A, Marcos R, Ortega JJ, et al. Breaks at telomeres and TRF2-independent end fusions in Fanconi anemia. Hum Mol Genet. 2002;11:439–444
14. Liang J, Yagasaki H, Kamachi Y, Hama A, Matsumoto K, Kato K, et al. Mutations in telomerase catalytic protein in Japanese children with aplastic anemia. Haematologica. 2006;91:656–658
15. Yamaguchi H, Baerlocher GM, Lansdorp PM, Chanock SJ, Nunez O, Sloand E, et al. Mutations of the human telomerase RNA gene (TERC) in aplastic anemia and myelodysplastic syndrome. Blood. 2003;102:916–918
16. Zhang A, Zheng C, Lindvall C, Hou M, Ekedahl J, Lewensohn R, et al. Frequent amplification of the telomerase reverse transcriptase gene in human tumors. Cancer Res. 2000;60:6230–6235
17. Von Zglinicki T. Oxidative stress shortens telomeres. Trends Biochem Sci. 2002;27:339–344

aplastic anemia; Fanconi anemia; telomeres; telomerase

© 2011 Medical Research Journal