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1 Department of Medical Technology, Ehime Prefectural University of Health Sciences, Tako-oda, Tobe, Ehime 791-2101, Japan
2 Department of Legal Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan
3 Laboratory of Molecular Biology, Kansai Medical University (Prof. Emeritus), Hirakata, Osaka 573-1136, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The X-inactivation pattern provided a good tool for tracing the origin and development of cell populations (especially malignant) without specific markers, when the subject is heterozygous for X-linked genes, and the paternal and the maternal alleles were distinguishable from each other (Fialkow 1973; Dow et al. 1985; Champion et al. 1997). One of the X-linked genes, the human androgen receptor gene (HUMARA), has highly polymorphic CAG trinucleotide repeats in the first exon (Edwards et al. 1992) and, therefore, many women are heterozygous for the HUMARA allele. The method named HUMARA assay (Allen et al. 1992), widely used for examination of X-inactivation patterns of various cell populations, utilizes this polymorphism and restriction enzymes that do not cut a methylated site.
The skin cells are considered to be distributed in patches on the outer surface of the body, each of the patches representing a clone descended from one of the epidermal progenitor cells. Recent two studies, both of which have compared the X-inactivation patterns of contiguous epidermal skin pieces from healthy women using the HUMARA assay, have indicated that normal human skin exhibits a fine mosaic of small patches in terms of the X-chromosomes (Asplund et al. 2001; Chaturvedi et al. 2002). On the other hand, linear patterns in the skin called the Blaschko's lines have been commonly observed in heterozygous X-linked gene defects, where it was assumed that clonality existed in the skin lesions (Happle 1985; Happle & Frosch 1985; Moss et al. 1993). But the area covered by a Blaschko's line is usually much larger than the size of patches observed in the two studies mentioned above.
Epidermal stem cells give rise not only to a wide range of skin types but also to various types of appendages (Byrne et al. 2003). Among appendages, the nail, which develops at specific sites, has been less intensively studied, with no previous data about the mosaic patterns in terms of the X-chromosomes. In this study, we have performed the HUMARA assay on DNA samples extracted from nail plates to investigate the X-inactivation patterns of the nail. The results suggested that the composition of precursor cells of each nail plate is maintained at each site over several cycles of regeneration time, and that each nail plate has a longitudinal linear band pattern, each band expressing only one of the two X-chromosomes. This is the first report which shows the pattern of lyonization in the normal human nail.
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Results |
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Figure 1 (1st and 3rd rows) show the representative X-inactivation patterns of finger and toe nails, respectively, from individual A. Table 1 summarizes the % inactivation of the longer allele in addition to the allele ratio of finger and toe nails from nine individuals. For any single individual, the X-inactivation patterns were different from nail to nail; in some nails either the longer or the shorter allele was exclusively inactivated, but in others two types of cells with either allele inactivated were present. Nails of the first type were found in seven individuals examined (A, B, C, D, G, H and I) , and were found at every position of both fingers and toes (Table 2). The frequency of fingers and toes of this type was 19.7% (13 out of 66) and 34.8% (16 out of 46), respectively, with no significant difference between finger and toe (
2 test with Yates’ correction). Among the five positions of both fingers and toes, the frequency of the nails of this type ranged from 14.3% (2 out of 14) (finger positions 1 and 4) to 44.4% (4 out of 9) (toe positions 3 and 4), but the difference between each position was statistically insignificant for both fingers and toes (2 test with Yates’ correction) (Table 2). Among total nail samples, the frequency of the nails of this type, that is, only expressing one of the two alleles, was 25.9% (29 out of 112).
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From individual A, right and left finger and right toe nail samples (from positions 2 to 5) were again obtained 2 years after the first experiment. Nails at position 1 were also again obtained, but these samples were used for the experiment in Fig. 2, as described below. The X-inactivation patterns of all samples in the second experiment (Fig. 1, 2nd and 4th rows) were the same as those obtained in the first experiment (Fig. 1, 1st and 3rd rows). The stability of the X-inactivation patterns indicates that the composition of precursor cells in the matrix, where the nail plate is produced continuously (Runne & Orfanos 1981), is constant for at least 2 years.
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Next, we examined in detail six nail samples from position 1 which were composed of two types of cells with almost equal allele ratio (an allele ratio of 1 : 1 to 2 : 1), by dividing each of them into three contiguous parts; radial (tibial) one-third, middle one-third and ulnar (fibular) one-third. If two types of cells with either the longer or the shorter allele inactivated were finely mixed, all parts should show identical inactivation pattern with an allele ratio of 1 : 1 to 2 : 1. As shown in Fig. 2, the inactivation pattern was different from part to part in each nail sample. In five out of six samples examined (finger L-1 and toe R-1 of individual A and finger R-1, finger L-1 and toe L-1 of individual C), one or two of the three parts showed extremely skewed X-inactivation pattern with an allele ratio more than 3, indicating that those parts were probably composed of either one type of cells, considering the inevitable fluctuation in the dividing lines. There were two nail samples in which the composition of two adjacent parts changed from one to another type of cells (toe R-1 of individual A and finger R-1 of individual C). These results suggest that most of the nails with inactivation pattern with an allele ratio between 1 : 1 and 2 : 1 are not a uniform fine mixture of two types of cells, but are composed of patches, in each of which only one of the two X chromosomes was inactivated.
Correlation of the X-inactivation patterns between two fingers or toes at different positions in the same hand or foot
The Pearson's correlation coefficients (r) between the % inactivation of nails at different positions with corresponding P value are shown in Table 3. Adjacent positions were more or less correlated, but the correlation between positions 2 and 3 was the lowest among such pairs and rather low. Positions farther apart were less closely correlated, except the remarkable strong correlation between 1 and 5.
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Discussion |
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As shown in Fig. 2, some nails are patchy mosaics of two types of cells with either the maternal or the paternal X-chromosome inactivated. The finding that there were two samples (toe R-1 of individual A and finger R-1 of individual C), in which the composition of two adjacent parts changed from one to another type of cells, suggests that other parts with almost equal allele ratio are probably not fine mixtures, but are also composed of (smaller) patches of two types of cells. Considering the constancy of the X-inactivation pattern of the nail through several cycles of regeneration time (Fig. 1) and the growth direction of the nail plate, that is, from proximal to distal in normal conditions (Runne & Orfanos 1981), we suggest that the nail plate has a longitudinal band pattern, usually with several bands, each band being composed of one type of cells. Since the width of these big nail samples was 12–14 mm and the length was about 10 mm, the width and the area of one-third of these nail plates should be 4–5 and 40–50 mm2, respectively. Even if considering the narrower bands, area size of bands occupied by cells expressing a single allele could be rather larger than the size of clonal unit of epidermis reported by Asplund et al. (2001) (20–350 basal cells) and Chaturvedi et al. (2002) (2 mm in diameter), and is smaller than the Blaschko's line. It is believed that dermis–epidermis interactions play essential roles in differentiation of epidermal stem cells into skin and various types of appendages and their proliferation (Parkinson 1992; Olivera-Martinez et al. 2004). Fibroblasts derived from various skin dermis at different anatomical sites have been shown to express different genes included in cell signaling pathways that control proliferation, cell migration and fate determination (Chang et al. 2002). These previous findings have indicated that, not only the destiny but also the clone size of the descendant cells from one epidermal stem cell may depend on where it is located. Thus, since different skin types and different appendages may have different sized mosaic patches in terms of X chromosomes, our findings and the hypothesis that the Blaschko's lines are clones derived from a stem cell, as well as the findings of Asplund et al. (2001) and Chaturvedi et al. (2002), may not be inconsistent with one another. However, the biopsy samples from the skin are usually accompanied by other types of cells, such as melanocytes or fibroblasts, as was indeed shown by Moss et al. (1993) in their cytogenetic studies. PCR assay is so sensitive that small number of contaminating cells could easily compromise the results. Therefore, conclusions using the biopsy skin samples should be viewed with caution. One of the advantages of using the nail as the sample is to avoid that problem and, moreover, nails can be obtained from restricted regions of the skin without inflicting any pain on the subject.
The allele ratio of the cell population reflects the mosaic cell composition (Fialkow 1973). We assumed that the mosaic cell composition of the nail is constant over time for the purpose of estimating how many progenitor cells gave rise to the nail plate during development, also assuming that the process follows the binominal distribution model. As there was no significant difference of the frequency of nails composed of one type of cells among the positions or between the finger and the toe (Table 2), we further assumed that the number of progenitor cells is the same for each finger and toe nail and, therefore, we considered all the nail samples as a homogenous population. If a nail were derived from a single cell with either X-chromosome inactivated, only the inactivation pattern with one of the two alleles exclusively inactivated should be found at all positions. The present results indicate that this is not the case. If there were n progenitor cells, the frequency of nails composed of one type of cells should depend on the probability of one X-chromosome being inactivated in any progenitor cell, and the nails from pn + qn of heterozygotes should show the inactivation pattern with one of the two alleles exclusively inactivated (allele ratio = ), and 1 – pn – qn should show the inactivation pattern composed of two alleles with various allele ratios, where p and q are the probability of one and the other X-chromosome is inactivated in any progenitor cell, respectively, and p + q = 1. We assumed that the probability p is 0.5 in the embryonic skin where progenitor cells of the nail begin to develop. Such assumption may be reasonable because inactivation of X chromosomes is a random process and it can be supposed that cells with the paternal X- and the maternal X-chromosome inactivated have no differences in the survival advantage in the normal human embryo. We calculated the expected frequencies of the nails with an allele ratio = as the function of the number n of progenitor cells by the formula pn + (1 – p)n with p = 0.5 (Fig. 3). Since the observed frequency of nails with an allele ratio 10, which should correspond to theoretical allele ratio = , was 25.9% , the number of progenitor cells of the nail plate at each site is estimated to be about 3.
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Experimental procedures |
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After informed consent in which the nature and possible consequences of the study were fully explained, nails of healthy female students and colleagues of the authors of this study were cut by nail clippers by themselves. All of the donors had been confirmed to have their androgen receptor alleles heterozygous from other prior experiments.
Extraction of DNA from nail samples
Nail samples for each finger or toe were pooled separately and stored at room temperature until sufficient amounts (more than 50 mg) were obtained. We numbered the positions of the big, the index, the middle, the fourth and the little finger or toe as 1, 2, 3, 4 and 5, respectively. We further denoted the big, the index, the middle, the fourth and the little finger or toe as R-1 to R-5, respectively, for the right limb and L-1 to L-5 for the left. From one individual (individual A), nail samples were obtained 2 years after the first experiment. In some experiments, R-1 or L-1 nail sample was divided evenly into three parts; the radial (tibial) one-third, the middle one-third and the ulnar (fibular) one-third. Each part of the nail was pooled separately and stored until sufficient amounts were obtained, as described above. DNA was extracted from nail samples according to Krskova-Honzatkova & Sieglova (2000) with modifications. Nail samples were placed in a 1.5-mL microtube and washed twice in TNE buffer (10 mM Tris–HCl, pH 8.0; 100 mM NaCl; 10 mM EDTA, pH 8.0) and then once in sterile water with vigorously mixing. The samples were then air-dried and were ground into fine particles using Multi-beads Shocker (YASUI KIKAI, Osaka, Japan). The particles were transferred into a 2.0-mL microtube and were incubated with 40 µL of 2.5 M dithiothreitol for 2 h at 56 °C, and then with 500 µL TNE buffer, in addition to 50 µL of 10% sodium dodecyl sulfate and 10 µL of 10 mg/mL proteinase K for another 48 h at 56 °C with constant shaking. If not dissolved completely even after the incubation, another 10 µL of 10 mg/mL proteinase K was added and the mixture was further incubated at 56 °C until it became clear (for 10–24 h). DNA was then purified using phenol extraction (3 times), phenol–chloroform extraction (twice) and then chloroform extraction (twice) followed by ethanol and sodium acetate precipitation. The pellet was washed with 70% ethanol, air dried and dissolved in sterile distilled water. The quality of extracted DNA was evaluated by 1.5% agarose gel electrophoresis using TBE buffer system (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.5), confirming that it contained genomic DNA, which was retained at the top of the gel, in addition to fragmented DNA. The average yield of DNA from a single nail sample (50–100 mg) was about 1–2 µg.
X-inactivation analysis
X-inactivation was analyzed using the HpaII site located near the highly polymorphic trinucleotide repeat in the HUMARA (Edwards et al. 1992) according to the method described by Allen et al. (1992) with slight modifications. For each DNA sample, two reactions were prepared in 200 µL-PCR tubes: in one, DNA (about 100–200 ng) was digested with 10 U HpaII (Boehringer Mannheim, Germany); in the other, DNA was incubated with the buffer (10 mM Tris–HCl, pH 7.5–10 mM MgCl2–1 mM dithioerythritol) with no enzyme. All reactions were carried out in 5 µL total volume, covered by 5 µL silicon oil, for 15 h at 37 °C. After digestion, reactions were terminated by incubating the mixture at 99 °C for 10 min. To this reaction tube, 45 µL of polymerase chain reaction (PCR) mixture (50 mM KCl–1.5 mM MgCl2–10 mM Tris–HCl, pH 9.0 containing 1.25 U Taq DNA polymerase and the two primers at a concentration of 0.5 µM) was added. The primer sequences were 5'-TCCAGAATCTGTTCCA GAGCGTGC-3' for the sense primer, and 5'-GCTGTGAAG GTTGCTGTTCCTCAT-3' for the anti-sense primer. Following an initial denaturation step for 5 min at 94°C, 30 amplification cycles were performed involving 60 s at 94 °C, 30 s at 62 °C and 30 s at 72 °C using PC707 programtemp-control system (ASTEC, Fukuoka, Japan). In the final cycle, extension at 72 °C was prolonged for 10 min. Four micro liters of the amplification product was then evaluated by electrophoresis through a GeneGel Excel 12.5/24 gel using a GenePhor DNA Separation System (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) and silver staining of the gel. Stained gels were scanned using Image Scanner ES-2000 (EPSON) and band intensities were quantitated by densitometric analysis using Quantity One software (pdi, Huntington Station, NY). The linear correlation between the band intensity and the amount of DNA loaded on the gel was confirmed using known amount of DNA samples (data not shown). In order to examine the efficiency of the HpaII enzymatic digestion, male DNA sample is included. In HpaII predigested male DNA sample, no amplification product should be produced, because male X-chromosome is active and the site is unmethylated so that it should be cleaved. However, predigested male DNA sample gave a band with about 10% intensity (average of five experiments) of that of undigested sample. Thus, the average efficiency of HpaII digestion in these experiments is 90%.
Calculations and statistical analyses
The percent inactivation was calculated according to Tonon et al. (1998): this was defined as the percentage of the longer allele (upper band) to the sum of both alleles in HpaII predigested samples, following normalization for band intensities in the undigested samples. In addition, the allele ratio, which is defined as the ratio between the two X-linked alleles in a given sample, was calculated by dividing the ratio between band intensities of HpaII predigested samples (upper band/lower band) by the same ratio in undigested samples. If the ratio was < 1, the reciprocal value was considered. For each DNA sample, results are reported as the mean value from two or more PCR analyses. When no nail samples were available or no informative PCR results were obtained, we denoted this fact with an open column or (nd) in Table 1, respectively. All statistical analyses were done using the JSTAT application software. The correlation of the % inactivation between two fingers or toes at different positions in the same individual was estimated by means of Pearson's correlation coefficient (r) with corresponding P value. 2 test with Yates’ correction was used to analyze the difference of the frequency of nails.
Criteria for clonality of the whole nail sample
Considering the efficiency of the HpaII digestion as 90%, an observed allele ratio 10 (observed % inactivation of the longer allele > 91% or < 9) corresponds to the cell population being composed of only one type of cells (theoretical allele ratio = ), and an observed allele ratio < 10 corresponds to a cell population being composed of two types of cells with various allele ratios.
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: mokada{at}epu.ac.jp
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References |
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Received: 23 July 2007
Accepted: 21 January 2008
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