In conclusion, we have extended our earlier observations of shortened telomeres in older premutation adults [Jenkins et al.., 2008a
] to a cohort of younger carriers (mean, 33.8 years; range, 8.6 to 56.2 years) as well as younger males (mean, 12.0 yrs; range 7.1 to 19.4) with full mutation alleles.
Six measures were used to demonstrate shorter telomeres in 8 of 8 male individuals with the FMR1
1) PNA telomere probe light intensities on chromosome 1 alone were determined.
2) Using the cen 2 ratio data provided a non-telomere standard that allowed us to generate light intensity ratios: telomere intensities/cen 2 intensities [Perner et al., 2003]. Although we obtained nearly the same results with and without the use of the cen 2 control, we recognize that light intensities can vary from preparation to preparation or from lot-to- lot of PNA probe, so that using the cen 2 probe, which is not expected to be affected by factors that are associated with telomere shortening - such as Alzheimer Disease [Panossian et la., 2003], degenerative defects of aging [Armanios et al., 2009], progerin production during cell senescence [Cao et al., 2011], and cellular aging [Mather et al., 2011] - allowed us to normalize the results.
3) Measuring the absolute telomere length in microns (micrometers) was done.
4) The same rationale for using cen 2 ratios was applied for using the absolute telomere length in microns/inter-telomere chromosome 1 length for analysis of telomere length of chromosome 1 in microns. Its application was even more theoretical than cen 2, because the t test p values for comparing telomere lengths alone or analyzing the ratio values were the same (p < 0.000001).
5) Determination of the mean number of chromosome arms with no signal and finding significantly more signal loss in premutations versus controls.
6) Finally, we utilized and compared reduced light intensities in interphase preparations.
Our interphase findings, in agreement with the others, allowed us to rely upon interphase preparations solely for light intensity comparisons in cells from male individuals with the full FMR1 mutation. This is important because the use of interphase preparations allows the most efficient use of time to complete the analysis. Use of interphase is less labor-intensive than using metaphase preparations and allows for increased productivity. However, we believe that interphase preparations provide results with increased variability due to increased background fluorescence, resulting in more “noise” in the data generated. In regard to reliability, all methods are appropriate. If interphase preparations are not used, then we recommend physical measurement of telomeres in microns as the next most productive and practical methodology since telomere and inter-telomere chromosome lengths may be analyzed without the use of an expensive image analyzer and it is faster than all other methods except interphase. Similarly, signal number loss can also be determined without the use of an image analyzer and accompanying expensive software, but it is the most labor intensive.
Finally, our observations suggest that telomere shortening in the premutation range is not a consequence of the development of FXTAS, since most individuals in the newer cohort are much younger than the age range for development of the neurodegenerative disorder. Telomere shortening could be due to pathogenic mechanisms that are active much earlier in the lives of premutation carriers, like the alterations in lamin A nuclear morphology [Garcia-Arocena et al., 2010
] that was also observed by Cao et al. 
during the induction of cell senescence. However, when mean light intensity interphase values from premutations were compared to those of the full mutation males, premutation T lymphocytes exhibited longer telomeres (premutation mean, 71.3 × 103
; full mutation mean, 45.8 × 103
; p <0.013) suggesting that something in full mutation individuals is causing even greater reduction in telomere length. Therefore, we suggest that telomere shortening may not be solely related to the effects of RNA toxicity, that are largely limited to the premutation range.