Instability analysis of the expanded GAAs in the <i>FXN</i> gene across different somatic tissues.

<p>Genomic DNA was extracted from heart (H), cerebral cortex (Cc), spinal cord (Sc), cerebellar cortex (Cb) and pancreas (P) tissues and the GAA repeats in the <i>FXN</i> locus were amplified by PCR. The results from FRDA patients (<b>A</b>) F2 (<b>B</b>) F7 and (<b>C</b>) M6 shown as examples. (-) represents no-template control and (+) represents positive control for amplification of the expanded GAAs (genomic DNA isolated from fibroblasts obtained from an unrelated FRDA patient). (<b>D-F</b>) A GAA repeat tract at the 5q23 locus was amplified by PCR using the same genomic DNA templates used for reactions shown in (<b>A-C</b>). (<b>G-I</b>) A fragment spanning intron 1—exon 2 of the <i>FXN</i> gene, downstream of the GAA tract, was also amplified using the same templates to serve as a control for genomic DNA quality.</p

Somatic instability of the expanded GAA repeats in Friedreich’s ataxia

<div><p>Friedreich’s ataxia (FRDA) is a genetic neurodegenerative disorder caused by transcriptional silencing of the <i>frataxin</i> gene (<i>FXN</i>) due to expansions of GAA repeats in intron 1. FRDA manifests with multiple symptoms, which may include ataxia, cardiomyopathy and diabetes mellitus. Expanded GAA tracts are genetically unstable, exhibiting both expansions and contractions. GAA length correlates with severity of FRDA symptoms and inversely with age of onset. Thus, tissue-specific somatic instability of long GAA repeats may be implicated in the development of symptoms and disease progression. Herein, we determined the extent of somatic instability of the GAA repeats in heart, cerebral cortex, spinal cord, cerebellar cortex, and pancreatic tissues from 15 FRDA patients. Results demonstrate differences in the lengths of the expanded GAAs among different tissues, with significantly longer GAA tracts detected in heart and pancreas than in other tissues. The expansion bias detected in heart and pancreas may contribute to disease onset and progression, making the mechanism of somatic instability an important target for therapy. Additionally, we detected significant differences in GAA tract lengths between lymphocytes and fibroblast pairs derived from 16 FRDA patients, with longer GAA tracts present in the lymphocytes. This result urges caution in direct comparisons of data obtained in these frequently used FRDA models. Furthermore, we conducted a longitudinal analysis of the GAA repeat length in lymphocytes collected over a span of 7–9 years and demonstrated progressive expansions of the GAAs with maximum gain of approximately 9 repeats per year. Continuous GAA expansions throughout the patient’s lifespan, as observed in FRDA lymphocytes, should be considered in clinical trial designs and data interpretation.</p></div

Determination of GAA repeat length in paired FRDA patient fibroblast (F) and lymphocyte (L) samples.

<p>(<b>A</b>) Agarose gel analysis of GAA repeat sizes in fibroblast/lymphocyte samples isolated from the same individual (FRDA patients FA1—FA16). (<b>B</b>) The mean sizes of the GAA1 and GAA2 alleles between all fibroblast and lymphocyte samples (n = 16) were calculated and compared. A p-value <0.05 denotes a significantly significant difference. (<b>C</b>, <b>D</b>) Correlation between the number of GAA repeats expanded in lymphocytes relative to size of the repeat tracts in fibroblasts and the number of GAA repeats in lymphocytes. The difference between the expanded GAA repeat lengths observed in lymphocyte and fibroblast samples (ΔGAA) was plotted against lymphocyte GAA sizes for each of the 16 FRDA paired samples. The analysis was performed for both alleles (<b>C</b>) GAA1 and (<b>D</b>) GAA2. The Pearson’s correlation coefficient (R) is indicated.</p

Assessing time-dependent changes of GAA tract length in FRDA lymphocytes.

<p>(<b>A</b>) The GAA repeat tract at the <i>FXN</i> locus was amplified using genomic DNA extracted from lymphocytes, which were isolated from 5 patients (F10, F11, F12, M7 and F13). Blood samples were taken at an initial timepoint (I) and a second time point 7–9 years after the initial sampling (II) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189990#pone.0189990.t003" target="_blank">Table 3</a>). The time-dependent changes in GAA repeat length were quantitated as (<b>B</b>) total GAA repeat gain/loss (ΔGAA) and (<b>C</b>) rate of the change (ΔGAA per year). (<b>D</b>) The Pearson’s correlation coefficient was calculated using the size of the GAA tract at time point I [GAA(I)] and the change of the number of GAAs between time points II and I [ΔGAA (II-I)]. Five pairs of samples (n = 10 alleles) were analyzed.</p

GAA repeat length in fibroblasts and lymphocytes of FRDA patients.

<p>GAA repeat length in fibroblasts and lymphocytes of FRDA patients.</p

Quantitative analysis of expanded GAA repeat instability in the <i>FXN</i> gene across different tissues.

<p>The expanded GAA repeats in the <i>FXN</i> gene were amplified from genomic DNA extracted from heart (H), cerebral cortex (Cc), spinal cord (Sc), cerebellar cortex (Cb) and pancreas (P) tissues isolated from FRDA patients. The band intensity of the PCR products and repeat size are shown for FRDA patients (<b>A</b>) F2, (<b>B</b>) F7, and (<b>C</b>) M6. Solid vertical lines represent the mean of all GAA repeat sizes detected across all five tissues for each patient. Gel lanes were manually outlined and gel bands were detected via the Image Lab 5.0’s band finder set to high sensitivity. Faint bands of PCR products not detected by the software were manually identified. Band boundaries, accounting for smearing, were automatically outlined by the program with final manual adjustments to include the entire spectrum of PCR products. Multiple PCR analyses with determinations of GAA lengths were performed to demonstrate reproducibility of PCR and reliability of measurements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189990#pone.0189990.s004" target="_blank">S4 Fig</a>).</p

Characterization of the FRDA patient tissues.

<p>Characterization of the FRDA patient tissues.</p

Characterization of FRDA patients.

<p>Characterization of FRDA patients.</p

Correlative analyses between mean GAA repeat sizes and patient age of death.

<p>The mean tissue GAA tract length in FRDA patient (<b>A</b>) heart, (<b>B</b>) cerebral cortex, (<b>C</b>) spinal cord, (<b>D</b>) cerebellar cortex, and (<b>E</b>) pancreas is plotted against the patient’s age at time of death. Statistical significance was reached for spinal cord, cerebellar cortex and pancreas (p <0.05).</p