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Cell cycle and centromere FISH studies in premature centromere division
© Corona-Rivera et al; licensee BioMed Central Ltd. 2005
Received: 09 March 2005
Accepted: 20 September 2005
Published: 20 September 2005
Mitotic configurations consistent in split centromeres and splayed chromatids in all or most of the chromosomes or premature centromere division (PCD) have been described in three categories. (1) Low frequency of PCD observed in colchicines-treated lymphocyte cultures from normal individuals. (2) High frequency of PCD with mosaic variegated aneuploidy. (3) High frequency of PCD as a sole chromosome abnormality observed in individuals with no recognizable clinical pattern. We report four members of a family with the third category of PCD.
Cell cycle duration assessed by average generation time using differential sister chromatid stain analysis and FISH studies of DNA centromere sequences in PCD individuals, are included and compared with previously reported PCD individuals from 9 families.
We observed PCD in colchicine-treated cultures from the propositus, his father, and two paternal aunts but not in his mother and four other paternal and maternal family members, as well as in untreated cultures from the propositus and his father. We observed cytological evidence of active centromeres by Cd stain. Significative cell cycle time reduction in anaphases of PCD individuals (average generation time of 21.8 h;SD 0.4) with respect to individuals without PCD (average generation time of 31.8 h;SD 3.9) was observed (P < 0.005, Student t-test for independent samples). Increased cell proliferation kinetics was observed in anaphasic cells of individuals with PCD, by differential sister chromatid stain analysis. FISH studies revealed the presence of alpha satellite DNA from chromosomes 1, 13, 21/18, X, all centromeres, and CENP-B box sequences in metaphasic and anaphasic cells from PCD individuals.
This report examines evidences of a functional relationship between PCD and cell cycle impairment. It seems that essential centromere integrity is present in these cases.
Mitotic configurations consistent in split centromeres and splayed chromatids in all or most of the chromosomes or "premature centromere division" (PCD), have been described in three categories. They are:
(2) High frequency of PCD (5% or more) with mosaic aneuploidies involving a variety of chromosomes, called "mosaic variegated aneuploidy", observed in individuals with microcephaly, growth deficiency, severe mental retardation, and risk of malignancy [3–5].
(3) High frequency of PCD (5% or more) as a sole chromosome abnormality [6–10]. Individuals with this condition have no recognizable clinical pattern or occur in healthy individuals. Association of this PCD trait with abortions and infertility has been reported [6, 7, 9, 10], but other authors suggest this PCD trait to be harmless [1, 8, 11].
We report a family that corresponds to the third category of PCD, in which four individuals showed PCD as a sole chromosome abnormality. Cell cycle duration, assessed by average generation time, and FISH studies of the centromere, are considered. Findings in the family are compared with those on 30 other previously reported individuals with PCD from 9 families. This report examines the evidences of a relationship between PCD and cell cycle impairment of cells bearing main structural centromere components from PCD individuals.
Percentages of PCD in repetitive colchicine-treated cultures from family members.
No. of cultures
Total mitoses scored
Paternal aunt (II-3)
Paternal aunt (II-8)
Paternal aunt (II-9)
Paternal aunt (II-10)
Maternal aunt (II-14)
Maternal aunt (II-15)
Cell cycle studies
Cellular proliferation kinetics was used to determine the cell cycle duration, which is the interval between one mitosis and the subsequent . The method called average generation time (AGT) by differential sister-chromatid stain [14, 15], was used to determine cell cycle durations. We compared the results between family members with more than 5% of PCD versus those with less than 3% of anaphase frequencies. The original AGT method [14, 15], consider for cell cycle calculations only metaphases, in this family, calculations were also performed considering, prophases, anaphases and total mitotic cells. The procedure was as follows. We obtained AGT's of each individual from accumulated data of 3 simultaneous 72 h cultures with 5 μg/ml of 5'-bromodeoxyuridine (SIGMA), added 24 h after set-up. In each culture, we scored prophases, metaphases and anaphases which had completed either one, two or three cell cycles identified by differential sister chromatid stain pattern. Then, AGT, were calculated per mitotic stage and per individual as follows. (i) Taking into account the percentage of cells at first (M1), second (M2) and third (M3) cell cycle, the replication index (RI) was obtained from this formula: RI = (1X%M1+2X%M2+3%M3)/100. (ii) The RI was used to obtain the AGT following the equation: AGT = time of harvest after exposure to 5'-BrdU/RI. Finally, AGT's were compared in paternal individuals with PCD versus those individuals with low anaphase frequencies from the maternal sibship. Statistical test t-Student for independent samples was used to compare both groups.
We searched for the presence of constitutive structural components of the centromere such as alpha satellite DNA sequences in the propositus and his parents with FISH according to a standard protocol , using alpha satellite DNA directly labeled probes to chromosomes 1, 13/21, 18, and X, as well as all centromeres probe. Besides, we searched for the presence of CENP-B box sequence in the propositus using a biotin labeled probe, following the protocol of Matera and Ward (1992) .
Cell cycle studies
Cell cycle durations in paternal sibship with PCD (paternal aunt II-8, propositus III-14, father II-11) versus maternal sibship without PCD (maternal aunts II-14 and II-15, mother II-12).
Average generation time
Paternal sibship with PCD Mean* (SD)**
Maternal sibship without PCD Mean* (SD)**
Comparison between groups t value
P < 0.005
We report four family members with more than 5% of colchicine-anaphase frequencies as a sole chromosome abnormality. Nine families have been reported referring to this trait as PCD [6–10]. Mitosis obtained from colchicine arrested lymphocyte cultures of normal individuals, show rates below 3% , 5%  or 1% observed in our control individuals. Previous reports of PCD frequencies without mosaic variegated aneuploidy (MVA), ranges 5 to 38% [6–10]. In our family, PCD was observed in the propositus, his father, and two paternal aunts in repeated colchicine-treated cultures in average frequencies of 7 to 22%. It was shown , that PCD can be induced through hypotonic increasing time treatment of mitotic cells in peripheral blood lymphocytes of healthy individuals and patients homozygous to PCD trait or PCD and MVA. They found that 0.075 M KCl at 37°C for 20 min, showed 0–2% cells in PCD which fits with our observed frequencies because 15 minutes of hypotonic treatment were used in our peripheral blood cultures. Total mitosis scored in repeated cultures of previous [6–10], and present report did not provide evidences of MVA. Although PCD is considered a rare phenomenon, two studies found in selected population frequencies of 1 of 100  or 1 of 1000 .
Main features of published families with PCD and present report.
Rudd et al. 1983 
Gabarrón et al. 1986 
Madan et al. 1987 
Bajnoczky and Gardó 1993 
Keser et al. 1996 
Individuals with PCD
Individuals with abortion or infertility
PCD in colchicine treated cultures *
PCD in untreated cultures *
Cell cycle progression requires control mechanisms that could be associated to PCD origin. A basic defect of cell cycle progression or metaphase-anaphase transition in PCD was suggested . Matsuura et al. (2000) , demonstrated that cultured fibroblasts from two infants with PCD and mosaic variegated aneuploidy are insensitive to the colcemid-induced mitotic-spindle checkpoint. Mitchel et al. (2001) , found that mitotic checkpoint defective MAD2+/- haploinsufficient human colon carcinoma cells showed 20% of precocious anaphases with prematurely separated sister chromatids, compared with 1% in wild-type cells, proposing that PCD is a suitable cytogenetic marker for the identification of mitotic checkpoint defects. In our family PCD was also observed in cultures without colchicine from the propositus and his father as in other PCD reports [6–8] (Table 3). Cultured PCD cells with defective colcemid-induced mitotic-spindle checkpoint reported by Matsuura et al. (2000) , were unresponsive to colchicine. Although in our family MVA was not observed as in Matsuura et al. (2000)  report, in both cases the mitotic arrest signal was overruled in PCD cells. Hanks et al. (2004) , provided the evidence that gene mutations can result in a defective spindle checkpoint in humans. They screened the full coding sequence and intron-exon boundaries of BUB1B, and found truncating and missense mutations inherited from different parents in five of eight families with mosaic variegated aneuploidy providing the first evidence in humans that gene mutations might be responsible for aneuploidy in human cancers. Interestingly, cytogenetic data of families 1, 4 and 5 in Hanks et al. (2004)  report showed also PCD, as well as in 6 of those 15 reported cases update . Considering that subjacent genetic cause was demonstrated to MVA, this opens the possibility that BUB1B defects can be involved in PCD origin. Mitotic spindle checkpoint serves as a surveillance mechanism that ensures the faithful transmission of chromosomes from a mother cell to its two daughter cells during mitosis , this can be involved also in PCD origin. On the other hand, because at least 6 genes have been involved with such checkpoint , the origin of PCD may be heterogeneous and should involve a mechanism that triggers the whole set chromosome segregation at mitotic spindle checkpoint.
Other aspect to be considered in PCD origin is the centromere. A basic defect of centromeric region in PCD was suggested . Two essential DNA sequences of the centromere were evaluated by FISH in this family: alpha satellite and CENPB-Box sequences. Centromere function requires the presence of alpha satellite DNA in all human centromeres . We observed alpha satellite DNA from all centromeres and the centromere of chromosomes 1, 13/21, 18, and X, as well as centromeric 17 bp CENPB-Box sequences in prophasic, metaphasic and anaphasic cells from the propositus and his parents. CENPB-Box sequence interacts with the kinetochore protein CENP-B required for the pairing of sister chromatids as structural support and in the conformation of primary constriction and kinetochore . Cytological evidence support the presence of functional centromeres in PCD cells, by positive Cd stain in the propositus and his parents and in one previous PCD report . Also, primary constrictions are evident in this and previous PCD reports. It seems that essential centromere integrity is present and remains unclear if whether or not is involved in PCD origin. Other mechanisms related to cell cycle regulation and functional components of the centromeric region such as defective centromeric cohesion , or kinetochore defective proteins are probable.
The PCD trait as a sole chromosome abnormality occurs in healthy individuals. Some authors suggest this PCD trait to be harmless [1, 8, 18]. In this report four individuals presented PCD, and three of them were phenotypically normal. Noteworthy, all the 30 PCD individuals from 9 previous families included in Table 3 were also phenotypically normal. Individuals with this category of PCD have no a recognizable clinical pattern [6–10]. In three of such families clinical findings were informed and reported as coincidental [6, 8, 10]. The abnormal phenotype observed in the propositus shows no concordance to previous PCD without MVA cases. PCD trait observed in healthy paternal relatives and all previous cases, represent the common one dose effect of this autosomal dominant trait (OMIM, *176430) . Such mode of inheritance was concordant with this report because male to male transmission was observed. In autosomal dominant PCD and abnormal phenotype associated to MVA, homozygosity was implicated [28, 29]. This statement was confirmed by Plaja et al. (2001)  in three patients compared with 8 previous cases exhibiting microcephaly, CNS anomalies, mental retardation, prenatal and postnatal growth retardation and cancer, proposing that in vivo occurrence of random aneuploidies and chromosome or genome instability disorder explained some of the clinical data. It seems that variegated aneuploidy is associated to an abnormal phenotype. Our propositus showed prenatal and postnatal growth retardation, profound developmental delay, hypoplasia of the brain and clonic seizures, coincident with MVA reports , but our case did not show MVA nor apparent cancer risk. Alternatively, considering that the inheritance of MVA is recessive , and some heterozygotes show levels of PCD without variegated aneuploidy, this can be compatible with those individuals described in present report or in previous reports regarding apparently harmless PCD. However, the relationship between PCD and MVA is uncertain . Also, we considered the possibility that neurogical affectation in the patient studied by us could be associated with neonatal hypoxia. In these cases, genetic heterogeneity may be involved.
Association of this PCD trait with abortions and infertility has been reported [6, 7, 9, 10]. This was observed in 12 of 34 PCD individuals from 8 of 10 previous families including present report (Table 3). We observed infertility in two paternal aunts (II-2 and II-3 in fig. 1); cytogenetical analysis was available only in one of them observing PCD. The estimated abortion frequency in descendents of reported PCD individuals was 37% (22 of 60) which is higher than those observed in general population of 15% . In one report, unexplained recurrent abortion observed in both parents with PCD was considered the consequence of abnormal behavior of the centromeres involving probable homozygous effect . Previous observations are coincidental but remark the occurrence of subfertility in PCD individuals.
Present report represents a new family with PCD as a sole chromosome abnormality. Cell cycle studies revealed that cell cycle reduction could be considered a distinctive finding in these cases. Based in previous reports and the fact that cells of PCD patients were unresponsive to colchicine is probable that a defective colcemid-induced mitotic-spindle checkpoint is involved. Is open the possibility that BUB1B defects or other genes involved in such checkpoint may be involved in PCD origin. In this cases considered DNA centromeric sequences were present. It seems that essential centromere integrity is present and remains unclear if whether or not is involved in PCD origin. Other mechanisms related to cell cycle regulation and functional components of the centromeric region may be involved. Interestingly, the PCD trait as a sole chromosome abnormality occurs in healthy individuals and there is not a characteristic associated abnormal phenotype. Only subfertility seems to be a common finding in these families. Those families deserve further investigation in order to understand possible mechanism of this mitotic trait.
We wish to thank Dr. Lisa G. Shaffer for her valuable comments, and laboratory facilities. We wish to thank to Dr. A. Baldini who kindly provided the CENP-B box biotin labeled probe. We are greatly indebted to Rogelio Troyo Sanromán by his statistical assistance and to Venancio Vazquez by his technical support. This work was supported by CONACYT M-5051 and Universidad de Guadalajara funds.
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