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Telomere lengths at birth in trisomies 18 and 21 measured by Q-FISH
Ken-ichi Nakamura a,⁎, Naoshi Ishikawa a,⁎, Naotaka Izumiyama a, Junko Aida a, Mie Kuroiwa b, Naoki Hiraishi c,Mutsunori Fujiwara d, Atsushi Nakao e, Tadashi Kawakami e, Steven S.S. Poon f, Masaaki Matsuura g,Motoji Sawabe h, Tomio Arai h, Kaiyo Takubo a,⁎a Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japanb Department of Pathophysiology, Yokohama College of Pharmacy, Yokohama 245-0066, Japanc Department of Laboratory Medicine, Hadano Red Cross Hospital, Hadano, Kanagawaken 257-0017, Japand Department of Pathology and Laboratory Medicine, Japanese Red Cross Medical Center, Tokyo, Japane Department of Neonatal Medicine, Japanese Red Cross Medical Center, Tokyo, Japanf Terry Fox Laboratory, British Columbia Cancer Research Centre, Vancouver, BC, Canadag Bioinformatics Group, Genome Center and Department of Cancer Genomics, The Cancer Institute, The Japanese Foundation for Cancer Research, Tokyo 135-8550, Japanh Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo 173-0015, Japan
Trisomies 18 and 21 are genetic disorders in which cells possess an extra copy of each of the relevant chromo-somes. Individuals with these disorders who survive birth generally have a shortened life expectancy. As telo-meres are known to play an important role in the maintenance of genomic integrity by protecting thechromosomal ends, we conducted a study to determine whether there are differences in telomere length atbirth between individuals with trisomy and diploidy, and between trisomic chromosomes and normal chromo-somes. We examined samples of peripheral blood lymphocytes (PBLs) from 31 live neonates (diploidy: 10, triso-my 18: 10, trisomy 21: 11) and estimated the telomere length of each chromosome arm using Q-FISH. Weobserved that the telomeres of trisomic chromosomes were neither shorter nor longer than the mean telomerelength of chromosomes as a whole among subjects with trisomies 18 and 21 (intra-cell comparison), and wewere unable to conclude that therewere differences in telomere length between 18 trisomy and diploid subjects,or between 21 trisomy and diploid subjects (inter-individual comparison). Although it has been reported thattelomeres are shorter in older individuals with trisomy 21 and show accelerated telomere shortening with age,our data suggest that patients with trisomies 18 and 21 may have comparably sized telomeres. Therefore, itwould be advisable for them to avoid lifestyle habits and characteristics such as obesity, cigarette smoking, chron-ic stress, and alcohol intake, which lead to marked telomere shortening.
About 2500 congenital human anomaly syndromes have been re-ported to date, among which the causative genes of about 400 havebeen identified and cloned (http://www.ncbi.nlm.nih.gov/omim/). Tri-somy 21 (Down syndrome) has one of the highest incidence ratesamong live neonates, and is also characterized by premature aging.Most of the affected patients manifest the various changes associatedwith normal aging at a relatively young age, and such changes includeincreased incidence rates of atherosclerotic heart disease, degenerative
bone and joint diseases, leukemia, and Alzheimer's disease (Wisniewskiet al., 1978). Schupf et al. (1994) found that women who bear childrenwith trisomy 21 before age 36 years have a high risk of developingAlzheimer's disease. The risk of a trisomy pregnancy is clearly relatedto higher maternal age (Hassold et al., 1985). Telomeres play criticalroles in themaintenance of chromosomal stability, as well as in limitingthe ultimate replication capacity of cells, and it has been suggested thattelomere shortening is an important biological factor in cell senescence,cell mortality, and aging (de Lange, 1998; Greider, 1998; Harley andVilleponteau, 1995). Blood lymphocytes of individuals with trisomy 21exhibit a 3-fold reduction of telomere length relative to those of normalindividuals in vivo (Vaziri et al., 1993), although telomere lengths in tri-somy 21 at birth have not been reported. Excessive telomere shorteningmay be attributable to a larger annual loss with more rapid turnoverand/or lower telomerase activity, or to the fact that telomeres are con-genitally shorter than those in controls. It is suggested that telomereshortening does not occur at the same rate for all telomeres, and infact, telomere shortening of specific chromosomes has been reported
Fig. 1. Relationship between fluorescence intensity demonstrated by Q-FISH and telomere restriction fragment (TRF) lengths in TIG-3 (32 and 74 PDL), TIG-103 (12 and 48 PDL), and TIG-114 (48 PDL) cells used for calibration. (A) Smears of TRFs forthe three TIG cell strains at five different PDLs by Southern blot analysis. (B)Median values of TRF length for thefive samples (measured using the “Telometric” software package). TRF lengths at 32 and 74 PDL (TIG-3), 12 and 48 PDL (TIG-103), and 48PDL (TIG-114)were 8.6, 5.2, 9.1, 6.9, and 6.0 kbp, respectively. (C) Frequency of telomere fluorescence units (TFUs) at five different PDLsmeasured using theQ-FISHmethod and TFL-Telo V2. Themedian values formetaphase spreads at 32 and 74 PDL(TIG-3), 12 and48 PDL (TIG-103), and 48 PDL (TIG-114)were 15,517 (15metaphase spreads), 5660 (10 spreads), 15,599 (11 spreads), 4929 (16 spreads), and 3843 (16 spreads) TFUs, respectively. (D) Linear relationship between TFUvalue estimatedby Q-FISH and TRF length measured by Southern blotting at the five different PDLs (R2 = 0.824, p = 0.033).
200K.N
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Fig. 1 (continued).
201K. Nakamura et al. / Gene 533 (2014) 199–207
in certain diseases (Rashid-Kolvear et al., 2007). Youngmothers of triso-my 21 children have been reported to have a normal telomere length(Dorland et al., 1998). We hypothesized that trisomy 21 childrenwould have a normal telomere length at birth, but that the telomeresof their trisomic chromosomes would be specifically shorter. To testthis hypothesis, we measured the individual telomere lengths on thep- and q-arms of all chromosomes in patients with trisomies 18 and21 using quantitative fluorescence in situ hybridization (Q-FISH) andcompared themwith telomere lengths in patientswith other conditionsand normal controls. We also compared the telomere lengths of triso-mic chromosomes with those of the other chromosomes in affected in-dividuals at birth.
2. Materials and methods
2.1. Subjects
Blood samples were obtained at birth from 31 babies suspected tohave chromosomal abnormalities, with informed consent from one orboth of the parents. Among these 31 subjects, 10 were diagnosedas having trisomies 18 and 11 as having trisomy 21; the remaining 10(diploidy group) lacked cytogenetically abnormal karyotypes. Samplesof peripheral blood were obtained within 7 days after birth in 27 sub-jects and at about one month after birth in 4.
Peripheral blood cells from the subjects were incubated for 72 h inRPMI 1640 medium supplemented with 20% calf serum and stimulatedwith phytohemagglutinin using amodified version of a technique report-ed previously (Moorhead et al., 1960). Phytohemagglutinin-stimulatedperipheral lymphocytes (preferentially T cells) were treated withcolcemid (0.1 mg/ml, Sigma Co., Rödermark, Germany) for 1 h, andfollowed by hypotonic shock and methanol/acetic acid fixation. Cellsuspensions were dropped onto clean glass slides and used forhybridization experiments.
2.1.1. Telomere length calibration (TRF vs. TFU)Calibration of telomere length was performed by comparison with
data obtained by Southern blotting and telomere fluorescence intensity(expressed as an arbitrary unit: the telomere fluorescence unit, TFU)determined by Q-FISH in 3 fibroblast strains at 5 different populationdoubling levels (PDLs).
2.1.2. Southern blot analysis of fibroblast strainsIn the fibroblast strains employed (Ohashi et al., 1980), i.e. TIG-3 (32
and 74PDLs), TIG-103 (12 and 48 PDLs), and TIG-114 (48 PDL), DNAdeg-radation was examined by pulse-field gel electrophoresis (Schwartz andCantor, 1984). We used the standard Southern method to measure
terminal restriction fragment (TRF) length with the restriction enzymeHinf I, and estimated the median representative values of TRF lengthusing a software package, Telometric (Grant et al., 2001).
2.1.3. Probes and counterstaining for calibrationThemetaphase chromosomes of the TIG cells and peripheral lympho-
cytes from the subjects were hybridized using the peptide nucleic acid-FISH preparation method (Poon and Lansdorp, 2001a,b). The Cy3-labeled (CCCTAA)3 peptide nucleic acid (PNA) probe (Telo C, cataloguenumber F1002; Fasmac, Atsugi, Japan) was used to label the telomeres,and a FITC-labeled CTTCGTTGGAAACGGGGT PNA probe (CENP1; a non-specific centromere probe, custom-made; Fasmac)was used for the cen-tromere. The chromosomeswere counterstainedwith 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR, USA).
2.1.4. Q-FISH and image analysis of fibroblast telomeresQ-FISH and image analysis were performed as described previously
(Poon and Lansdorp, 2001a; Takubo et al., 2010). The fluorescence in-tensities of telomeres were analyzed with the TFL-Telo V2 softwarepackage (Terry Fox Laboratory, BC Cancer Research Centre, Canada). Atotal of 10 to 16metaphase spreads for each of the three TIG cell strains(five samples)were analyzed. Telomere intensities of individual arms inthe metaphase spreads were measured. For calibration, the relativeTFUs were then extrapolated from, and correlated with Southern blotdata for the three TIG cell strains (five PDLs).
2.2. Karyotype analysis of the subjects
For chromosomal analysis of lymphocytes, the metaphase spreadswere karyotyped from air-dried FISH samples (DAPI-chromosomesand FITC-centromeres), and also by the standard air-drying method(Seabright, 1971). The metaphase spreads were analyzed using theISIS Karyotyping System (Metasystem GmbH, Altlussheim, Germany)(Supplementary data, Figs. S1A, B, C). Two of the authors who were ex-perts in cytogenetics (MF, KN) then checked and confirmed the karyo-types. A total of 10 or 11 metaphases from each of the diploid, trisomy18, and trisomy 21 samples were analyzed cytogenetically and countedto determine the modal number.
2.2.1. Telomere measurement by Q-FISHWemeasured the lengths of 92 (diploidy) and94 (trisomy) individual
telomeres situated on the p- and q-armsof eachmetaphase chromosome,aswell as the telomere lengths of controlfibroblasts.Measurementswereobtained from at least 10 metaphase spreads for each subject. Each sub-ject group (diploidy, 18 trisomy, and 21 trisomy) was analyzed se-quentially. We calculated telomere lengths on the basis of a linearrelationship (y = 0.00026x + 4.82, as mentioned in the Results sec-tion). For each subject, we calculated the mean telomere lengths forthe p- and q-arms of each chromosome to determine if there wereany chromosome arms with specifically short telomeres. In addition,we compared the mean telomere lengths for the individual p- and q-arms with those of chromosomes 18 and 21 in the subjects withtrisomy.
2.2.2. Telomere length calibration with TIG-1A set of samples for diploid (SN2935), 18 trisomy (SN0185), 21 triso-
my (SN2533) and control cell strain TIG-1 (32PDL) were applied onthree slides (first slide: SN2935/SN0185/SN2533, second slide: TIG-1/SN2935/SN0185, third slide: TIG-1/SN2935/SN2533) and processed bya single Q-FISH procedure using the same materials and within thesame vessels.
2.3. Statistical analysis
In the calibration experiment, differences in p values were analyzedusing Welch's t test for independence. Fisher's Z test was used for
0
5
10
15
20
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X0
5
10
15
20
Tel
omer
e Le
ngth
(kb
p)
A. Diploidy(Case 2992-18, Thanatophoric Dysplasia)
Fig. 2. Representative FISH images of lymphocytes from normal diploid individuals, and patients with trisomy 18 and trisomy 21, along with the telomere length for each arm.(A) Karyotype of a lymphocyte from a patient with thanatophoric dysplasia (ID: 2992, Table 2). Left: a metaphase spread; middle: after karyotyping of the left-hand image;right: telomere lengths for each p-arm and q-arm from this case, respectively. Mean telomere lengths for all the p-arms and q-arms were 8.1 ± 2.0 and 8.1 ± 2.1 kbp, respec-tively. (B) Karyotype of a lymphocyte from a patient with trisomy 18 (ID: 0045, Table 3). Left: a metaphase spread; middle: after karyotyping of the left-hand image; right:telomere lengths for each p-arm and q-arm from this case, respectively. Mean telomere lengths for all the p-arms and q-arms were 9.1 ± 2.2 and 9.0 ± 2.0 kbp, respectively.(C) Karyotype of a lymphocyte from a patient with trisomy 21 (ID: 1001, Table 4). Left: a metaphase spread; middle: after karyotyping of the left-hand image; right: telomerelengths for each p-arm and q-arm from this case, respectively. Mean telomere lengths for all the p-arms and q-arms were 9.1 ± 2.2 and 8.7 ± 1.8 kbp, respectively.
202 K. Nakamura et al. / Gene 533 (2014) 199–207
comparison of relationships. The telomere lengths for the p- and q-arms were compared within individual cases using the t test. Thetelomere lengths for the p- and q-arms within each group were com-pared using the paired t test. The telomere lengths for the p- and q-arms on different chromosomes were compared using the t test.For all comparisons, differences at p b 0.05 were considered to besignificant.
3. Results
3.1. Calibration
3.1.1. Southern blot analysis of fibroblastsAll DNA samples examined by pulse-field gel electrophoresis
were more than 100 kbp in size. The telomere lengths (median
values as representative) at the five PDLs were 8.6 (TIG-3, 32 PDL),5.2 (TIG-3, 74 PDL), 9.1 (TIG-103, 12 PDL), 6.9 (TIG-103, 48 PDL),and 6.0 kbp (TIG-114, 48 PDL) (Figs. 1A, B).
3.1.2. Relationship of telomere fluorescence intensity in the TIG strains atdifferent PDLs
The Q-FISH measurements of the same five PDLs as those used forSouthern analysis are shown in Fig. 1C. The median values calculatedas representative for the karyotyped cells at the five PDLs were:15,517 TFUs (median for 15 metaphase spreads, TIG-3, 32 PDL), 5660TFUs (10 spreads, TIG-3, 74 PDL), 15,599 TFUs (11spread, TIG-103, 12PDL), 4929 TFUs (16 spreads, TIG-103, 48 PDL), and 3843 TFUs (16spreads, TIG-114, 48 PDL).
Scatter-plot analysis demonstrated a robust relationship betweenthe TRF and TFU values at the five PDLs. As shown in Fig. 1D, a linear re-lationship (y = 0.00026x + 4.82) was demonstrated.
3.1.3. Calibration of representative cases of diploidy, 18 trisomy and 21trisomy with TIG-1
This calibration experiment indicated that serial experiments usingthe same materials and procedures would provide comparable data,and that additional calibration using a single cell strain (TIG-1 at 32PDL) as a standard confirmed the robustness of inter-experimentalcomparison. However, we were unable to perform such calibrationwith TIG-1 for every subject because of the limited amount of the sam-ple in some cases (Supplementary data; Supplementary Table, Table S1,Supplementary Figure, Fig. S2).
3.2. Mean telomere lengths for the p-arm, q-arm, and both in the threegroups
FISH images of metaphase spreads were obtained for all 31 subjects,and these revealed heterogeneity of telomere lengths for the p- and q-armswithin any givenmetaphase spread (Figs. 2A, B, C; Supplementarydata, Figs. S3A, B, C). A total of 883 metaphase spreads were examinedand karyotyped. Mean telomere lengths for the p-arm, q-arm, and
both, in patients with diploidy, trisomy 18, and trisomy 21 are shownin Table 1. Mean telomere lengths for the diploidy, trisomy 18, and tri-somy 21 groups were 8.2 ± 2.6, 8.4 ± 2.0 and 9.1 ± 2.7 kbp, respec-tively. (The mean telomere lengths for the three groups calibratedusing TIG-1were 10.7, 9.5 and 10.1 kbp, respectively.) The standard de-viations (SD) of telomere lengthwere large. In this experiment, wewereunable to find any marked differences of whole telomere length amongthe three groups. However, since calibration using a single cell strain(Sections 2.2.2 and 3.1.3) could not be performed in every set of exper-iments, the statistical significance of inter-group comparisons remainedunclear.
Mean telomere lengths for the chromosome arms overall are shownin Fig. 3. In chromosomes 11, 12, 16, 17, and 19, the mean length of thep-arms was less than that of the mean value of p-arms overall, and themean length of the q-armof chromosome20was less than that of the q-arms overall in the three groups.
3.3. Telomere lengths within each individual subject in the diploidy group
Table 2 shows the mean telomere lengths for the p-arm, the q-arm,and both, in each patient in the diploidy group. Three of the 10 subjectsshowed significantly longer telomeres in the p-arms than in the q-arms.Paired t-test showed that telomere lengths for the p-arms were signifi-cantly (p = 0.001) greater than for the q-arms in the diploidy group asa whole.
3.4. Telomere lengths within each individual in the trisomy 18 group
We found that telomeres of trisomic chromosomeswere not shorterthan themean telomere length of chromosomes as awhole among sub-jects with trisomy 18 (intra-cell comparison, shown in Table 3, eighthcolumn).
In 3 of the 10 subjects with trisomy 18, themean telomere length forthe p-arm was significantly greater than that for the q-arm (Table 3,fifth column). However, paired t test showed that the telomere lengthfor the p-arm was not significantly different from that of the q-arm inthis group as a whole. The mean telomere length for the p- or q-armof chromosome 18 was significantly greater in 3 subjects, and was lessin 3 subjects, than the mean length for the p- or q-arm among the 47arms. In this group overall, paired t test showed that the telomere lengthof the q-arm (p = 0.001) for chromosome 18 was significantly longerthan those of the 47 chromosome p-arms as a whole.
3.5. Telomere length within each individual in the trisomy 21 group
As was the case in the trisomy 18 group, the telomeres of trisomicchromosomeswere not shorter than themean telomere length of chro-mosomes as a whole among subjects with trisomy 21 (intra-cell com-parison, shown in Table 4, eighth column).
In 5 of the 11 subjects with trisomy 21, themean telomere length forthe p-arm was significantly greater than that for the q-arm (Table 4).Paired t test showed that the telomere length for the p-arm was signif-icantly greater (p = 0.008) than that for the q-arm in the group as awhole.
The mean telomere length for the p- or q-arm of chromosome 21was significantly greater in 4 subjects, and was less in 3 subjects, thanthe mean length for the p- or q-arm among the 47 arms. Amongthe cases in this group, paired t test showed that the telomere of thep-arm of chromosome 21 was longer than the telomeres of all the p-arms (p = 0.011).
4. Discussion
Telomere lengths in normal adult bone marrow metaphases are re-portedly very heterogeneous, as was also reflected in our results, where-as the telomere lengths of individual chromosomes are comparable and
Blue shows significantly longer. Red shows significantly shorter.
, , : Commonly shorter in the 3 groups.
Fig. 3.Mean telomere lengths for the p- and q-arms of lymphocytes from normal diploid individuals, and patients with trisomy 18 and trisomy 21. In chromosomes 11, 12, 16, 17, and 19,the mean length of the p-arm was less than that for all 46 or 47 p-arms, and themean length of the q-arm on chromosome 20 was less than that for all of the q-arms in the three samplegroups.
204 K. Nakamura et al. / Gene 533 (2014) 199–207
significantly better correlated between sister chromatid pairs thanbetween telomeres on opposite ends of individual chromosomes(Lansdorp et al., 1996). As has been shown in yeast, where loss of a singletelomere results in cell-cycle arrest and chromosome loss (Sandell andZakian, 1993), individual telomeres may mediate important biologicaleffects.
On the basis of regression analysis, two classical papers have report-ed that TRF length is reduced significantly by 27, 31 and 33 bp per yearas a function of age in normal human blood cells (Hastie et al., 1990,Slagboom et al., 1994). More recently, Son et al. (2000) reported thatthe telomere length of peripheral blood CD4+, CD 8+ and CD19+lymphocytes at birth was within 10 kbp, and showed a year-by-yearreduction with age thereafter. The first telomere length quantification
of 21 trisomy patients was reported by Vaziri et al. (1993). They foundthat the yearly TRF length reduction rate in blood lymphocytes was41 bp in normal subjects and 133 bp in patients with trisomy 21, sug-gesting that accelerated telomere loss is a biomarker of prematureimmunosenescence in patients with trisomy 21. Flow FISH analysis oftelomere length in lymphocytes from individuals with trisomy 21(n = 19, 18–60 years) has shown that the mean telomere length wasreduced to about 47% over the course of 40 years from 20 to 60 yearsof age (Brando et al., 2004). These papers indicated that telomere reduc-tion in trisomy 21 was very rapid in comparison with controls.
With regard to the telomere length at birth, an initial report byHastie et al. (1990) indicated that themean TRF length in PBLs fromnor-mal individuals could be extrapolated from a regression line to around
Table 2Mean telomere lengths (kbp) in peripheral lymphocytes at birth in the diploidy group.
Cases Sex Diagnosis (chromosome with causative gene) No. of
telomere
signals
Mean
telomere
length
Mean
telomere
lengths for
p-arm
Mean
telomere
length for
q-arm
p-Value
(All p vs
All q)
2421 F Incontinentia pigmenti (Xp11.21 or Xq28) 2024 9.3±2.6 9.4±2.7 9.2±2.6 0.150
2992 F Thanatophoric dysplasia (4p16.3) 1840 8.1±2.0 8.1±2.0 8.1±2.1 1
2460 M Hydrocephalus and myelocystomenigocele 1840 7.3±1.4 7.3±1.4 7.2±1.3 0.160
2469 M Tetralogy of Fallot 1840 10.3±3.3 10.4±3.4 10.1±3.1 0.032
2935 M Congenital atresia of the esophagus 1840 8.5±2.3 8.6±2.6 8.4±2.2 0.183
2986 F Fever and hyperpnea 1840 8.3±2.3 8.5±2.4 8.2±2.3 0.034
3035 M Complete transposition of great arteries 1839 6.4±1.2 6.4±1.2 6.4±1.1 0.247
3270 M Hypospadias and premature birth 1840 9.6±3.1 9.7±3.1 9.4±3.2 0.035
3276 F Tetralogy of Fallot 1840 6.8±1.3 6.8±1.3 6.7±1.3 0.148
3355 F Patent truncus arteriosus 1840 7.3±1.8 7.3±1.8 7.4±1.7 0.111
Mean
M 91998.4±2.8
(9199)8.5±2.9 8.3±2.7 < 0.001
F 93848.0±2.3
(9384)8.0±2.3 7.9±2.2 0.031
M
& F18583
8.2±2.6
(18583)8.3±2.6 8.1±2.5 < 0.001
Blue indicates significantly longer.Red indicates significantly shorter.M: male, F: female
205K. Nakamura et al. / Gene 533 (2014) 199–207
9.8 kbp at birth. Son et al. (2000) investigated the yearly reduction oftelomere length in peripheral blood CD4+, CD 8+ and CD19+ cellsby flow FISH, and reported that the mean telomere lengths at birthcould be extrapolated to 9.1, 7.0, and 7.5 for CD4+ and CD8+ T cellsand B cells, respectively. However, the report by Vaziri et al. (1993)lacked data for newborn individuals. If the expected telomere lengthin trisomy 21 individuals at birth could be extrapolated from the datathey obtained by regression analysis, it would be around 10.5 kbp, i.e.about 1 kbp greater than that of their control individuals and also indi-viduals reported previously. Recently, telomere shortening associatedwith an increase in the copy number of the telomerase RNA componentgene (TERC) in amniocytes of trisomy 21 fetuses has been reported(Sukenik-Halevy et al., 2011). These two major observations seem tobe contradictory, and are also discrepant with our data. This may be at-tributable to the different cell lineages examined in each study. Anothercritical differencewas the patient age (i.e. gestational stage).While all ofthe subjects in their study were fetuses sampled after pregnancy termi-nation, all of the patients in our study had been born naturally. There-fore, their life stages were completely different. Accordingly, the dataare insufficient for establishing a consensus. However, at least with re-gard to telomere lengths in PBS, the classical TRF data and our datastrongly suggest that telomere length at birth in individuals with 21 tri-somy may correspond to that of normal diploid individuals, and thatthereafter telomere erosion may accelerate at some point during devel-opment and/or growing stage.
A study of 76 metaphase cells from 17 humans aged 4 to 45 yearshas shown that telomere length was correlated with the size of the as-sociated chromosome arm (Wise et al., 2009). We also found heteroge-neity of telomere length in the p- and q-arms of 47 chromosomes in
subjects with trisomies 18 and 21 and 46 chromosomes in diploid sub-jects at birth. Unlike other studies that had shown telomeres on the q-arms to be longer, our study indicated that the mean telomere lengthson all p-arms were significantly longer than those on all q-arm in thediploidy and trisomy 21 groups, whereas the corresponding values intrisomy 18 were similar. These data suggest that telomere length regu-lation may be influenced by topological effects.
To address the above hypothesis, we examined whether or not telo-mereswere shortened in specific chromosomes.Wewere unable tofindany evidence for universal chromosome-specific telomere shortening,including trisomic chromosomes. However, we found a tendency fortelomere shortening in some chromosomes; for example, 11p, 12p,16p, 17p, and 19p were significantly shorter than the mean p-armlength, and 20q was significantly shorter than mean q-arm length. Pre-viously, in CD34+ cells purified from normal bone marrow of 16 indi-viduals aged 20 to 70 years, telomeres on chromosome 17p weresignificantly shorter than the median telomere length for all chromo-somes (Martens et al., 1998). In the present study, the mean telomerelength for chromosome 17p was also less than that for chromosomesoverall. Therefore, the telomeres on chromosome 17p may haveshown congenital shortening (Rashid-Kolvear et al., 2007). Furtherstudies will be necessary to clarify the degree of telomere length short-ening on specific chromosomes. In the present study, the telomeres oftrisomic chromosomes were not shorter than those of any other chro-mosomes among subjects with trisomies 18 and 21.
Patients with trisomy 21may develop Alzheimer's disease inmiddleage or older adulthood. Jenkins et al. (2006) have found that T lympho-cytes in individuals with both trisomy 21 and dementia have shortertelomeres than those in trisomy 21 patients without dementia. Trisomy
Table 3Mean telomere lengths (kbp) in peripheral lymphocytes at birth in trisomy 18 group.
Cases Sex No. oftelomere
signals
Meantelomere
length
Meantelomerelength for
p-arm
Meantelomerelength for
q-arm
p-value(all p vs All q)
Mean telomerelength for
chromosome 18p-value
(all vs 18 pq)
Mean telomerelength for p-armof chromosome
18p-value
(all p vs 18p)
Mean telomerelength for q-armof chromosome
18p-value
(all q vs 18q)
0002 M 2812 7.7±1.7 7.7±1.9 7.7±1.6 0.8788.0±1.7
0.022
7.7±1.5
1
8.4±1.7
< 0.0 01
0045 F 2820 9.0±2.1 9.1±2.2 9.0±2.0 0.139.2±1.9
0.175
9.0±2.0
0.674
9.4±1.8
0.065
0185 M 2824 8.5±1.9 8.4±1.9 8.5±1.8 0.5718.5±1.9
1
8.3±1.5
0.548
8.7±2.2
0.401
0802 M 2820 8.3±1.6 8.3±1.6 8.3±1.7 0.4998.2±1.7
0.418
7.6±1.4
< 0.001
8.7±1.9
0.032
2194 M 2820 7.9±1.7 7.8±1.7 8.0±1.8 0.0028.1±1.8
0.127
7.7±1.3
0.490
8.4±2.1
0.080
2407 M 3004 8.1±1.7 8.2±1.8 8.0±1.6 < 0.0018.1±1.7
1
8.2±1.6
1
8.0±1.8
1
2922 F 3179 8.0±1.8 8.0±1.8 8.0±1.7 0.2618.1±1.8
0.442
8.2±2.0
0.280
8.0±1.6
1
2998 F 2820 7.4±1.4 7.4±1.4 7.3±1.4 0.6927.3±1.3
0.351
7.1±1.2
0.025
7.4±1.4
0.511
3124 F 3570 9.3±2.1 9.5±2.2 9.2±2.0 < 0.0019.1±1.9
0.127
9.4±2.1
0.637
8.8±1.8
0.038
3130 F 3760 9.3±2.1 9.5±2.3 9.2±1.9 < 0.0019.4±1.9
0.433
9.2±1.8
0.0 84
9.6±1.9
0.026
Mean
M 14280 8.1±1.8 8.1±1.8 8.1±1.7 18.2±1.8
0.104
7.9±1.5
0.007
8.5±2.0
< 0.001
F 16149 8.7±2.1 8.7±2.2 8.6±2.0 0.0028.7±1.9
1
8.7±2.0
1
8.7±1.9
0.267
M &
F30429 8.4±2.0 8.4±2.0 8.4±1.9 1
8.4±1.9
1
8.3±1.8
0.095
8.6±1.9
0.001
Blue indicates significantly longer.Red indicates significantly shorter.M: male, F: female
206 K. Nakamura et al. / Gene 533 (2014) 199–207
21 also results in immune dysfunction, including thymus abnormalitiesand premature aging of immune cells. The accelerated loss of telomeresin peripheral blood cells might reflect generalized early senescence ofimmune cells in these individuals. Previouslywe reported that telomerelengths in liver, kidney, brain, and myocardium in any given individualare significantly correlated, and that telomere length differs in a tissue-specific manner (Takubo et al., 2002). In view of the early onset ofsymptoms in trisomy 21 patients, it would be important to clarify therelationship between telomere length in affected organs and that in pe-ripheral blood cells.
As patients with trisomy 21 do not have shorter telomeres at birthand show accelerated telomere shortening in early life, it would be ben-eficial for them to avoid lifestyle habits and characteristics such as obe-sity, cigarette smoking (Valdes et al., 2005), excessive chronic stress(Epel et al., 2004), and excessive alcohol intake (Aida et al., 2011),which lead to marked telomere shortening. This might allow them tominimize the occurrence of diseases attributable to telomere-inducedchromosomal instability, and to live longer, healthier lives.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2013.09.086.
Conflict of interest
The authors declare that they have no competing interests.
Acknowledgments
The authors wish to thank Ms. K. Kanno, Mr. S. Nakazawa, Mr. N.Hosokai for helpful assistance with karyotyping, and Dr. D. Douglas for
language editing. This study was supported by Grants-in-Aid for Scien-tific Research from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (KT, Nos. B17390108, B21390109; KN: No.C18590354).
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Table 4Mean telomere lengths (kbp) in peripheral lymphocytes at birth in trisomy 21 group.
1001 M 2067 8.9±2.0 9.1±2.2 8 .7±1.8 < 0.0019.2±2.1
0.0969.7±2.3
0.0328.6±1.7
0.661
1002 M 2023 7.6±1.7 7.5±1.7 7.6±1.7 0.0687.7±1.8
0.5177.9±1.9
0.0587.5±1.7
0.646
1004 M 2249 12.4±3.3 12.4±3.4 12.4±3.2 112.7±3.4
0.29311.8±3.2
0.14813.6±3.4
0.002
2207 M 1868 11.2±2.8 11.2±3.0 11.1±2.7 0.44911.0±3.5
0.54112.2±4.2
0.0749.8±1.9< 0.001
2260 F 1813 8.0±1.9 8.1±1.9 7.9±1.8 0.0228.2±2.1
0.2818.7±2.5
0.0837.7±1.5
0.339
2533 M 1877 8.5±2.1 8.6±2.1 8.4±2.1 0.0398.6±2.4
0.6579.3±2.6
0.0458.0±1.9
0.151
2662 M 1880 9.1±2.0 9.1±2.0 9.1±1.9 19.3±2.0
0.2889.0±2.1
0.7089.5±2.0
0.115
2788 F 1859 7.4±1.6 7.5±1.6 7.4±1.5 0.1657.3±1.7
0.5127.7±2.0
0.4516.9±1.2
0.004
3110 F 1872 8.9±1.9 9.1±2.1 8.7±1.8 < 0.0018.8±2.3
0.6439.3±2.7
0.5758.3±1.7
0.097
3181 F 1877 8.6±2.1 8.7±2.4 8.4±1.9 0.0039.1±2.2
0.0129.3±2.3
0.0609.0±2.1
0.019
3396 F 1877 8.8±2.3 8.9±2.3 8.7±2.2 0.0548.5±2.4
0.1679.0±3.0
0.8018.0±1.6
0.002
Mean
M 11962 9.7±3.0 9.7±3.0 9.6±2.9 0.0649.8±3.1
0.37310.0±3.2
0.0849.6±3.0
1
F 9297 8.3±2.0 8.5±2.2 8.2±1.9 < 0.0018.4±2.2
18.8±2.5
0.0448.0±1.8
0.078
M & F
21259 9.1±2.7 9.2±2.8 9.0±2.6 < 0.0019.2±2.9
0.2339.5±3.0
0.0118.9±2.7
0.333
Cases Sex No. oftelomere
signals
Meantelomere
length
Meantelomerelength for
p-arm
Meantelomerelength for
q-arm
p-value(all p vs all q)
Mean telomerelength for
chromosome 21p-value
(all vs 21 pq)
Mean telomerelength for p-armof chromosome
21p-value
(all p vs 21p)
Mean telomerelength for q-armof chromosome
21p-value
(all q vs 21q)
Blue indicates significantly longer.Red indicates significantly shorter.M: male, F: female
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