Can Telomere Shortening in Human Peripheral Blood Leukocytes Serve as a Disease Biomarker of Friedreich's Ataxia?

Telomere Length and Diseases

T elomeres are protein–DNA complexes localized at chromosomal ends. They consist of highly conserved hexanucleotide repeats (TTAGGG) and play an essential role in maintenance of genomic stability. Telomere length (TL) shortens in each somatic cell division, because the 3′-DNA replication strand is incomplete while a ribonucleoprotein enzyme called telomerase reverse transcriptase mediates the synthesis of telomere repeats, balancing the natural chromosome erosion. The balance between these two contrasting forces controls telomere size. Aberration of the telomere complex does not allow the protection of the chromosome ends, leading to cellular senescence or apoptosis (9).

TL seems to be a variable trait with a genetic component, displaying high variations at birth among different populations. The telomere is longer in women than in men and undergoes progressive shortening with age. Several nongenetic factors such as obesity, smoking, and sedentary living also seem to play a role in TL (9). Oxidative stress has been proposed to be an important modulator of TL (8).

In humans, shortened TL is associated with rare monogenic diseases, including dyskeratosis congenita, aplastic anemia, idiopathic pulmonary fibrosis, and ataxia telangiectasia (1). Many studies have also reported the association between TL (with or without modulation of telomerase activity) and complex diseases such as type 1 and 2 diabetes, hypertension, cardiovascular disease, psychiatric disorders, and cancer (9). TL shortening has also been found in several age-related neurological diseases (Alzheimer, Parkinson, cerebral autosomal-dominant arteriopathy with subcortical infarcts, and leucoencephalopathy (CADASIL) and dementia after stroke) (9,6).

Friedreich's ataxia (FRDA; OMIM# 229300), the most common inherited ataxia, is an autosomal recessive disease that generally begins at puberty and leads to death in the fifth or sixth decade of life. FRDA is caused by a GAA-triplet repeat expansion in the first intron of the frataxin gene. This expansion impairs the transcription of the gene, leading to deficiency of frataxin. This protein is a ubiquitous mitochondrial protein involved in mitochondrial iron transport and/or in the assembly of iron–sulfur cluster enzymes (4). Frataxin deficiency is associated with iron dysregulation and with a severe disruption of iron–sulfur cluster proteins. Lack of iron detoxification and the increase in bioavailable iron are likely to enhance cellular sensitivity to oxidative stress (4) and activate inflammatory pathways (3). Therefore, we hypothesized that TL might be shortened in patients with FRDA.

Does TL of Peripheral Blood Leukocytes Correlate with FRDA?

To answer to this question, we have investigated TL in peripheral leukocytes from patients with FRDA and healthy controls. Sample details are shown in Table 1. There were more men than women in the control group; however, this difference was not statistically significant.

We found significantly shortened TL in patients compared with the controls (p=0.032, Fig. 1). Since TL is also influenced by age and gender, we employed a binary logistic regression model to take into account the role of these confounding variables. The difference in TL means remained statistically significant (p=0.012). In a subsample of patients, we also investigated the correlation between specific clinical features and TL. To this aim, we employed the Pearson test between TL and age, gender, GAA repeats, age of onset, and disease duration. As expected, there was a significant correlation between TL and age; TL was longer in younger patients (r=− 0.286, p=0.014). Gender was not associated with TL in leucocytes in either the healthy controls or the patients. There was no correlation between TL and the smaller expanded allele GAA1 and the larger expanded allele GAA2 repeats. Interestingly, we found a significant correlation between TL and disease duration. The patients with longer disease duration had shorter TLs (r=− 0.594, p=0.001) (Fig. 2). This finding remained significant after correction for age (p=0.012) or for age of onset (p=0.001) of FRDA. We were unable to analyze the possible relationship between TL and cardiomyopathy and/or diabetes because of the small size of these specific subsets of patients that were available.

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FIG. 1.

Box-plot and whisker diagram displaying the telomere length distributions in controls and patients with Friedreich's Ataxia (FRDA). Telomere length (TL) is expressed as a relative telomere length (T/S, see NOTES). Bold lines in the boxes indicate the median T/S values, and the lower and upper boundaries of the boxes are the first and third quartiles, respectively (p=0.032). The horizontal lines (whiskers) indicate the minimum and maximum T/S values.

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FIG. 2.

Scatter plot showing the negative correlation between T/S and disease duration in 27 patients with FRDA (r=− 0.594, p=0.001).

Could TL Be a Blood Biomarker in FRDA?

Strategies for FRDA treatment are currently under evaluation and several of them (antioxidant and iron chelators) are already in phases II and III of the process. To assess the efficacy of these therapeutic agents, different clinical and biochemical markers are used (4).

ICARS (International Cooperative Ataxia Rating Scale) and FARS (Friedreich Ataxia Rating Scale) are the most used clinical markers. It should be underlined that although these scales are useful for monitoring disease progression, they are not always appropriate for clinical trials (7). Biomarkers of oxidative stress have been discovered in urine (8-hydroxy-2′-deoxyguanosine) and blood (malondialdehyde) samples from patients with FRDA, but some conflicting results have been obtained, probably because of technical difficulties (4).

As already mentioned, FRDA is caused by decreased frataxin in mitochondria, which in turn results in mitochondrial dysfunction and oxidative damage in patients. The exact pathophysiology of FRDA is still unknown; however, a decrease in frataxin increases the amount of reactive oxygen species, making the cells more sensitive to oxidative damage (4). Frataxin deficiency has been reported previously to induce inflammatory genes (3).

Oxidative stress and inflammation are widely considered to be two main mediators of chronic diseases and are reported to increase the rate of leukocyte telomere attrition (9). How oxidative stress influences TL has not yet been fully elucidated. It has been suggested that because of the high guanine content in their sequences, telomeres are remarkably sensitive to damage by oxidative stress. Thus, enhanced oxidative stress and neuroinflammation could synergize telomere shortening in FRDA.

Here we report the first study of TL changes in patients with FRDA. The TL in patients with FRDA was significantly shorter than in the controls. We did not find any significant correlation between TL, GAA expansion, and age of onset. Interestingly, we did find a correlation between TL and disease duration. Patients affected by FRDA for a longer time showed more shortened TL also after adjustment by age. This finding may suggest that TL could be considered a biomarker of disease progression rather than disease severity.

The pathogenesis of TL shortening in FRDA needs to be further explored. It is possible to speculate that this phenomenon could depend on the increased oxidative stress and inflammation observed in patients with FRDA.

Limitations of the Study

A limitation of our study was the relatively small sample size, which reduced the statistical power needed to analyze comorbidities such as diabetes and cardiomyopathy. FRDA is a rare disease, and larger cohorts in cooperative studies will be needed to address this issue. These larger cohorts could also allow the study of other FRDA subphenotypes, such as late-onset and retained reflexes in patients.

Conclusions

Up to now, there is no effective treatment and no well-defined clinical or biochemical marker for FRDA. However, several trials are currently active. The improved understanding of the role of frataxin has led to the common use of antioxidants in FRDA clinical practice. It has been demonstrated that antioxidants decrease telomere shortening (4). Telomere measurement is increasingly used in other diseases as a reliable method to predict disease prognosis (5). Our data suggest that telomere size evaluation could be integrated with other clinical and biochemical markers to evaluate both disease progression and possible efficacy of antioxidant therapies.

Notes