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Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring
Mutation Research 520 (2002) 25–37
Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring Sara Frias a,∗ , Sandra Ramos a , Bertha Molina a , Victoria del Castillo a , Dora Gilda Mayén b a
Laboratorio de Citogenética, Departamento de lnvestigación en Genética Humana, Instituto Nacional de Pediatr´ıa, Insurgentes Sur 3700-C, Col. Insurgentes-Cuicuilco, Mexico DF, CP 04530, Mexico b Servicio de Genética, Instituto Nacional de Perinatolog´ıa, Mexico DF, CP 04530, Mexico Received 10 January 2002; received in revised form 19 June 2002; accepted 19 June 2002
Abstract Down syndrome (DS) resulting from free trisomy 21 (FT21) has been largely associated with advanced maternal age. However, approximately 60% of FT21 cases are born to young couples. Thus, the etiological factors responsible for these FT21 children must differ from those proposed for maternal age-related FT21. These factors have not been defined. In this study, we analyzed the chromosomes of peripheral blood lymphocytes from three groups of couples aged ≤35 years, to identify chromosomal trisomies: Group I included 5 couples with normal offspring; Group II included 22 couples with one FT21 child; and Group III consisted of 3 couples with recurrent FT21. A total of 13,809 metaphases were analyzed with G-banding and 60,205 metaphases were analyzed with FISH using a 13/21 centromeric probe. Aneuploidy was significantly more frequent in Groups II and III. The frequencies of hyperdiploid cells were 0.19, 0.49 and 0.96% in Groups I–III, respectively. FISH analysis showed that trisomy 21 cell percentages were 0.08, 0.21 and 0.76 for Groups I–III, respectively, and were very similar to those obtained with G-banding. Trisomy 21 mosaicism was found in 2/22 couples with one FT21 offspring, and in 2/3 couples with recurrent FT21. Our data suggest that mosaicism is an important cause of FT21 offspring in young couples, and that aneuploidy is more frequent among couples with FT21 offspring. This may be related with age and other undetermined intrinsic and extrinsic factors. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Trisomy 21; Parental mosaicism; Aneuploidy; Lymphocytes; Down syndrome; Etiology of trisomy 21
1. Introduction Down syndrome (DS) is the most frequent chromosomal disorder in humans, occurring in 1 of every 700 live births [1]. DS has five cytogenetic variants: (a) free trisomy 21 (FT21), defined as the presence of an extra chromosome 21 (FT21); (b) translocations involving a rearrangement of one chromosome 21 with ∗ Corresponding author. Tel.: +82-5255-5606-9580; fax: +82-5255-5606-9455. E-mail address: [email protected] (S. Frias).
a D- or a G-Group chromosome; (c) 21q21q isochromosome; (d) cellular mosaicism, defined as the presence of two different cell lines in the same individual, generally one normal and one trisomic line in variable proportions; and (e) partial trisomy of the 21q22.3 region [2–4]. In 95% of FT21 cases, the additional chromosome 21 is of maternal origin, and the remainder 5% is of paternal origin. Two of every 10 maternal origin cases and 3 of every 4 paternal origin cases, originate from non-disjunction during meiosis II [2–7]. While maternal origin FT21 is clearly correlated with advanced
1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 2 ) 0 0 1 6 3 - 8
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S. Frias et al. / Mutation Research 520 (2002) 25–37
age [1,8,9], paternal origin FT21 is not age-related [7]. However, although women above age 35 years have a higher risk of having FT21 offspring, <5% of all FT21 births occur to these women. Approximately 60% of FT21 cases are born to young couples [9,10]. Thus, the etiological factors of the latter and paternal origin FT21 must differ from those proposed for maternal age-related FT21 (for review, see [6,11]), and have not been clearly defined. Parental mosaicism is one of the main causes of recurrent FT21, and is also a cause of FT21 in young couples. Mosaicism is not exclusive to DS patients, as two different cell lines may be present in phenotypically normal individuals with a low frequency of trisomic cells. The abnormal cell line may be confined to one or more specific tissues, such as germ or blood cells, with no phenotypic consequences. This anomaly may remain unnoticed even after routine cytogenetic diagnosis, when the number of cells analyzed is insufficient to identify abnormal lines present in <10% of cells, and because of the preferential proliferation of normal as compared to trisomic lines [12]. In addition, if one of the parents has cryptic trisomy 21 mosaicism in the germ cells, he or she could be phenotypically normal, have a normal karyotype in lymphocytes, but have an increased risk of having FT21 offspring. There are many reports of recurrent FT21 infants born to mosaic parents [13–29]. Parental mosaicism as a cause of FT21 has been examined in few studies, with only 20–200 cells being analyzed [22,30]. We, therefore, investigated the presence of trisomy 21 mosaicism in parents of FT21 offspring performing chromosome analysis on a large number of peripheral blood lymphocyte metaphases by classical GTG-banding and fluorescence in situ hybridization (FISH). This enabled us to detect low levels of parental mosaicism as part of the etiology of FT21 in young couples.
2. Materials and methods 2.1. Subjects The study population consisted of Mexican couples with one or more FT21 offspring. All couples attended the Genetics Outpatient Services of the National
Institute of Pediatrics and the National Institute of Perinatology of Mexico from 1997 to 1999. To avoid advanced maternal age-related bias, we included only couples whose ages were ≤35 years at the time their FT21 offspring were born. Five volunteer Mexican couples aged ≤35 years with healthy offspring were included as a control group. All individuals were asked to complete a questionnaire that included family history and pedigree. Couples with a history of previous chemotherapy and/or radiotherapy or who had a viral infection within 1 week before sampling were excluded. A code was assigned to each couple. Parents of healthy offspring were coded as C1–C5 followed by Fa for ‘father’ or Mo for ‘mother’. Parents of FT21 offspring were coded as P1–P25; P22–P25 were recurrent FT21 cases. 2.2. Cell cultures and cytogenetic procedures Heparinized whole blood samples (0.5 ml) were drawn from all subjects, and cultured in 5 ml modified McCoy 5a medium (Gibco, USA) containing 0.25 ml phytohemagglutinin (Gibco, USA) and 0.1 ml reconstituted penicillin–streptomycin (Gibco, USA). Cultures were incubated at 37 ◦ C and harvested after 72 h using 0.1 ug/ml of colcemid (Gibco, USA) for 1 h, according to the standard method described by Moorehead et al. [31]. Cells were dropped on cold slides and air-dried to be processed for both classical and molecular cytogenetic studies. For GTG-banding, slides were treated with 0.015% trypsin (Gibco, USA) and 0.01% EDTA (Sigma, USA) in phosphate buffer (pH7). Chromosomes were stained with 5% Giemsa (Gibco, USA) in phosphate buffer. All slides were coded by an independent individual to ensure blind scoring. FISH was performed using a digoxigenin-labeled centrometric probe for chromosomes 13/21 (D13Z1/ D21Z1, Oncor kit, Gaithersburg, MD, USA). Chromosomes were denatured with 70% formamide with 2XSSC at 72 ◦ C for 2 min, and hybridized at 37 ◦ C for 24 h. Slides were washed with 0.2XSSC solution at 72 ◦ C for 2–5 min; chromosomes were incubated with fluorescein-labeled anti-digoxigenin antibodies (Oncor kit, Gaithersburg, MD, USA) for 30 min at 37 ◦ C and washed three times with 0.5% Tween 20 and 4XSSC for 2 min. Chromosomes were
S. Frias et al. / Mutation Research 520 (2002) 25–37
then counterstained with propidium iodine (Oncor kit, Gaithersburg, MD, USA). 2.3. Chromosome analysis The classical cytogenetic analyses included: (a) karyotype analysis of 15 GTG-banded metaphases per individual, examining the number and structure of all chromosome regions and subregions to identify constitutional aberrations; and (b) analysis of 200–400 GTG-banded non-adjacent metaphases to identify numerical chromosomal abnormalities [32]. In one couple (P2) only 190 and 165 metaphases per individual could be analyzed due to technical problems with the lymphocyte cultures. Whenever an aneuploid metaphase was identified, the chromosome number and chromatin condensation of all metaphases within a radius equivalent to the 10× objective lens field were also analyzed, in order to rule out false gain of chromosomes caused by technical artifacts. Technical artifact-related aneuploid metaphases were not considered for the analysis. For FISH analysis, metaphases were examined on an Olympus BX40 fluorescence microscope using triple band filters for DAPI, FITC and Texas red, and single filters for FITC and Texas red. At least 1000 metaphases per individual were scored in 57 parents, while only 965, 957 and 852 cells were analyzed in the three remainder. Fluorescent signals observed exactly on chromosome centromeres of similar size and intensity were considered as positive. Metaphases showing four signals (two on G chromosomes and two on D chromosomes) were considered as normal. Metaphases were considered as trisomic for chromosome 21 when three signals were observed on G chromosomes and two signals were observed on D chromosomes. Because our purpose was to identify mosaicism for a trisomic cell line, only trisomies and not monosomies were included for the analysis. Furthermore, an undetermined percentage of monosomies may be caused by technical artifacts such as broken metaphases or lack of hybridization. 2.4. Definition of mosaicism An individual was defined as a true trisomy 21 mosaic only if: (a) classical cytogenetics showed more
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than two cells exhibiting the same extra chromosome in the same sample [33,34] and (b) FISH analysis showed a frequency of trisomic cells greater than the mean percentage plus twice the standard deviation of the control group [35]. The power for detecting mosaicism according with the number of lymphocytes analyzed was: for a 95% confidence level when n = 200 cells are analyzed, the percent of mosaicism excluded is 2%; when n ≥ 400 cells are analyzed, the percent of mosaicism excluded is 1% [32]. Differences in the frequency of abnormal cells among the three groups were compared using the chi-square proportions test.
3. Results 3.1. Study population A total of 30 couples (60 individuals) aged ≤35 years were included: five with only normal offspring (Group I), and 25 with FT21 offspring. Twenty-two of these couples had only one FT21 child (Group II), and the remainder three couples had more than one FT21 child (recurrent DS, Group III). Background and family history of all participants are described in Tables 1–3. All couples belonging to Groups I and III, and 17/22 couples from Group II were Mexico City metropolitan zone (MMZ) residents. Mean parental age was 28.3, 26.2 and 25.7 years for Groups I–III, respectively. DS cases were the first child born to 11/22 couples in Group II and 2/3 couples in Group III. An excess of male DS cases was found, as the male/female sex ratio was 2.1 (19 males/9 females). 3.2. Classical cytogenetics Karyotypes were normal in all parents, as no constitutional (congenital) numerical or structural aberrations were found. However, constitutional chromosomal polymorphisms (normal variants) were observed in eight individuals (Table 4). Overall, 67 hyperdiploid cells were found among a total of 13,809 GTG banded cells (0.49%) distributed among all groups (Table 5). Supernumerary marker chromosomes (chromosomes resembling the
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S. Frias et al. / Mutation Research 520 (2002) 25–37
Table 1 Family background (Group I) Family code
Age (years)
Profession
Other data
Pregnancies/miscarriagesa
C1
Fa: 25 Mo: 20
Student Student
MMZ
1/0
C2
Fa: 30
MMZ
2/0
Mo: 30
Biologist working at beer factory Laboratory worker
Fa: 32
Administrative
MMZ, father smokes >5 cigarettes per day
2/0
Mo: 33
Laboratory worker
C4
Fa: 30 Mo: 29
Electronic engineer Computer engineer
MMZ
1/0
C5
Fa: 28 Mo: 26
Chauffer Housewife
MMZ Both parents smoke >5 cigarettes per day
3/0 (two induced abortions)
C3
Mean age: 28.3, S.D.: 38
Total pregnancies/miscarriages: 9/0
Age: at the time of birth of the last offspring; Fa: father; Mo: mother; MMZ: Mexico City metropolitan zone residents. a Only considered in miscarriages those spontaneous and not induced abortions. Table 2 Family background (Group II) Family code/sex of FT21 offspring
Age (years)
Profession
Other data
Pregnancies/miscarriagesa
P1/male
Fa: 18 Mo: 15
Merchant Housewife
MMZ
1/0
P2/male
Fa: 26
Builder
MMZ, chickenpox at week 13 of pregnancy
4/2
Mo: 27
Housewife
P3/male
Fa: 28 Mo: 21
Agronomy technician Housewife
MMZ
2/0
P4/female
Fa: 35
Lawyer
MMZ, asthmatic
5/3, one of the miscarriage products had a neural tube defect
Mo: 33
Housewife
P5/male
Fa: 23 Mo: 21
Carpenter Housewife
MMZ
2/0
P6/male
Fa: 25 Mo: 24
Office employee Housewife
MMZ
1/0
P7/male
Fa: 27 Mo: 26
Airline worker Housewife
Baja California Sur
2/0
P8/female
Fa: 34 Mo: 32
Copper plant worker Housewife
MMZ
1/0
P9/female
Fa: 35
Merchant
MMZ, father smokes 20 cigarettes per day, mother smokes 2 cigarettes per day
4/1
Mo: 34
Housewife
P10/male
Fa: 21 Mo: 21
Hardware employee Housewife
MMZ
1/0
S. Frias et al. / Mutation Research 520 (2002) 25–37
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Table 2 (Continued) Family code/sex of FT21 offspring
Age (years)
Profession
Other data
Pregnancies/miscarriagesa
P11/male
Fa: 27 Mo: 27
Not available Housewife
Chihuahua chih.
1/0
P12/male
Fa: 35 Mo: 33
Restaurant administrator Housewife
Veracruz ver.
2/0
P13/male
Fa: 28 Mo: 25
Grape cutter Housewife
Cuautla, mor.
3/0 (two induced abortions)
P14/female
Fa: 33 Mo: 30
Builder Housewife
MMZ
1/0
P15/male
Fa: 18 Mo: 16
Chauffer Housewife
MMZ
3/1
P16/male
Fa: 27 Mo: 26
Artisan (rug weaver) Housewife
Oaxaca oax.
3/1
P17/male
Fa: 20 Mo: 21
Waiter Housewife
MMZ
3/0
P18/male
Fa: 32 Mo: 30
Chauffeur Housewife
MMZ
3/0
P19/female
Fa: 33 Mo: 24
Data Non available
MMZ
1/0
P20/female
Fa: 27 Mo: 23
Carpenter Housewife
MMZ
1/0
P21/female
Fa: 16 Mo: 17
Storage employee Student
MMZ
2/0 (one induced abortion)
P22/male
Fa: 29 Mo: 27
Window cleaner Housewife
MMZ
1/0
Total male/ female: 15/7
Mean age: 26.2, S.D.: 5.62
Total pregnancies/miscarriages: 47/8 = 17% fetal lost
Age: at birth of the DS child; Fa: father; Mo: mother; MMZ: Mexico City metropolitan zone residents. a Only considered in miscarriages those spontaneous and not induced abortions. Table 3 Family background (Group III) Family code/sex of FT21 offspring
Age (years)
Profession
Relevant clinical data
Pregnancies/miscarriagesa
P23/two males
Fa: 25
Merchant
MMZ, asthmatic
3/1 (one elective abortion of a FT21 product)
Mo: 28
Housewife
Fa: 26
Builder
MMZ, one stillborn malformed child
4/0
Mo: 25
Housewife
P25/one male, one female
Fa: 25 Mo: 25
Grocery store employee Housewife
MMZ
5/2 (one stillborn)
Total male/female: 4/2
Mean age: 25.66, S.D.: 1.21
P24/one male, one female
Total pregnancies/miscarriages: 12/3 = 25% fetal lost
Age: at birth of the youngest DS offspring; Fa: father; Mo: mother; MMZ: Mexico City metropolitan zone residents. a Only considered in miscarriages those spontaneous and not induced abortions.
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S. Frias et al. / Mutation Research 520 (2002) 25–37
Table 4 Chromosomal polymorphisms in parents of normal and FT21 offspring Family code/father or mother
Karyotypea
I
C3/Fa
46,XY,22pstk−
II
P4/Mo P10/Mo P15/Fa P15/Mo P17/Fa P18/Mo P20/Fa
46,XX,inv(9) 46,XX,22pstk+ 46,XYqh+ 46,XX,21pstk+ 46,XY,inv(9) 46,XX,16qh+ 46,XYqh+
Group
No polymorphisms were observed among individuals belonging to Group III. a Notations: pstk−: decrease in length of the satellite stalk on the short arm; pstk+: increase in length of the satellite stalk on the short arm; qh+: increase in length of the heterochromatin on the long arm; inv(9): inversion of the chromosome 9.
morphology of a C–E or G chromosome, but not definitely identified) were found in 12 individuals (Table 5). Twelve of the 22 supernumerary markers were G-like, but did not involve chromosome 21 rearrangements as no extra 21 signal was observed by FISH in any case. The analysis of 200 or more GTG metaphases per individual revealed the presence of low percentages of trisomic cells in individuals from all three study groups. Trisomies involving normal chromosomes (non-markers) other than 21 were found in 11 individuals: 1 from Group I, 9 from Group II and 1 from Group III (Table 5). Only one trisomic cell involving chromosomes 10, 13, 17, 20, 22 or X was found in one individual from Group I, seven from Group II and one from Group III; while more than one trisomic cell was found in only two individuals: the asthmatic mother from Group II (P4/Mo) who had several aneuploid metaphases (two cells with trisomy 9, one cell with trisomy 17, and seven cells with trisomy 21); and one mother from Group II (P8/Mo) with three trisomic 47,XXX cells, representing a clone. A unique trisomic X cell was found in two more mothers; and trisomy X was the most frequent non-21 chromosome trisomy (Table 5). Chromosome 21 was more involved in mitotic non-disjunction than any other chromosome: 30/67 (44.7%) hyperdiploid cells had an extra chromosome 21. In Group I, 4/2200 (0.19%) metaphases were hyperdiploid, and trisomy 21 was found in only one.
In Group II, 51/10,355 (0.49%) metaphases were hyperdiploid, and 22 showed an additional 21 chromosome. In Group III, 12/1254 (0.96%) metaphases were hyperdiploid, and 7 had trisomy 21 (Tables 5 and 6). Overall, hyperdiploid metaphases and specifically trisomy 21 metaphases were significantly less frequent in Group I as compared to the groups with FT21 offspring (P < 0.05). Although mean age was similar in all groups, we assessed the effect of parental age by comparing the frequency of hyperdiploid metaphases in individuals aged 15–24 years and those aged 25–35 years. This sub-classification was feasible only in Group II, which included a sufficient number of subjects and showed the widest age dispersion (Table 1). Group IIa included 16 parents aged 15–24 years, and Group IIb included 28 parents aged 25–35 years (Table 6). The frequency of hyperdiploid and trisomy 21 cells was significantly higher in Group IIb than in Group IIa (P < 0.05) Moreover, even though the ages were similar the frequency of hyperdiploid cells was significantly higher (P < 0.05) in Group IIb than in Group I, mean age 28.3 years (only one mother aged <25 years). Although trisomy 21 cells were found in 17/60 individuals from all three groups, in most cases only one affected cell was identified. More than two trisomic 21 cells were found in 2/44 individuals belonging to Group II and in 2/6 individuals belonging to Group III (Table 5). 3.3. Molecular cytogenetics In accordance with the observations made by classical cytogenetics, FISH analysis revealed that chromosome 21 was significantly more involved in mitotic non-disjunction than chromosome 13. While only two individuals from Group II (P7/Mo and P18/Fa) showed one trisomy 13 cell, several individuals from all groups showed one trisomic 21 cell. Moreover, 11/44 subjects from Group II and 4/6 from Group III showed trisomy 21 frequencies above the threshold for mosaicism (0.3%). Percentages of trisomic 21 cells were 0.08, 0.21 and 0.76 for Groups I–III, respectively (Table 7). Tetraploid cells were identified in all groups, and the frequency of these cells was not significantly different among groups. FISH analysis revealed that total genome hyperdiploidy percentages calculated as described by Elhajouji [36] were 0.9, 2.5
S. Frias et al. / Mutation Research 520 (2002) 25–37
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Table 5 Frequency of trisomic cells in parents of normal and FT21 offspring, with hyperdiploid cells Group Family code/ father or mother I
II
III
Total
#Metaphases Number (%) of trisomic cells for individual chromosomes 9
C2/Fa C3/Mo C5/Fa Total Group Ia
200 200 200 2200
P1/Mo P2/Mo P3/Fa P3/Mo P4/Fa P4/Mo P5/Fa P7/Fa P7/Mo P8/Fa P8/Mo P10/Fa P10/Mo P12/Fa P12/Mo P13/Fa P13/Mo P16/Fa P17/Fa P17/Mo P18/Fa P18/Mo P19/Fa P19/Mo P21/Mo P22/Mo Total Group IIa
200 165 400 400
P23/Fa P24/Fa P24/Mo P25/Fa P25/Mo Total Group IIIa
400 400 200 200 200 200 200 200 200 200 300 300 200 200 200 200 200 200 200 200 200 10355 200 230 224 200 200 1254 13809b
10
13
17
20
21
22
X
Markers
Total
1 (0.5) 1 (0.5)
1 2 1 4
(0.5) (1.0) (0.5) (0.19)
2 1 2 (0.5) 3 3 (0.75) 3 2 10 1 1 (0.5) 2 1 1 3 1 1 1 (0.5) 1 1 1 (0.3) 2 1 (0.3) 1 1 (0.5) 2 1 1 1 1 3 1 1 4 (2.0) 4 51
(1.0) (0.6) (0.75) (0.75) (0.5) (2.5) (0.25) (1.0) (0.5) (0.5) (1.5) (0.5) (0.5) (0.5) (0.5) (0.6) (0.3) (1.0) (0.5) (0.5) (0.5) (0.5) (1.5) (0.5) (0.5) (2.0) (0.49)
1 3 3 4 1 12
(0.5) (1.3) (1.3) (2.0) (0.5) (0.96)
1 (0.5) 1 (0.5)
2 (1.0)
1 (0.6) 1 (0.25)
2 (0.5)
2 7 1 1 1 1
1 (0.25)
(0.5) (1.75) (0.25) (0.5) (0.5) (0.5) 3 (1.5) 1 (0.5) 1 (0.5)
1 (0.5) 1 (0.3) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 3 (1.5) 1 (0.5) 1 (0.5)
1 (0.5) 3 (1.3) 3 (1.3) 1 (0.5)
3 (1.5) 1 (0.5)
2 1 1 2 (0.014) (0.007) (0.007) (0.014)
1 30 (0.007) (0.21)
3 5 22 (0.021) (0.036) (0.15)b
67 (0.49)
Only parents and chromosomes involved in hyperdiploidy are shown. a Total metaphases analyzed per group. b Total metaphases in 60 individuals from all groups. Aneuploidy frequencies for specific chromosomes were estimated based on this number.
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S. Frias et al. / Mutation Research 520 (2002) 25–37
Table 6 Comparison of hyperdiploid cell percentages observed with G-banding (according to age groups) Group
n
Age (years)
Total spreads
Cells with trisomy 21 (%)
Cells with non-21 trisomy (%)
Total hyperdiploids (%)
I IIa IIb II III
10 16 28 44 6
25–35 15–24 25–35 15–35 25–35
2200 4000 6355 10355 1254
1 2 20 22 7
3 10 19 29 5
4 12 39 51 12
(0.05) (0.05) (0.3)a (0.21)a (0.56)a
(0.14) (0.25) (0.3)a (0.28)a (0.40)a
(0.19) (0.30) (0.6)a (0.49)a (0.96)a
Groups I and III include 25–35-year-old individuals, except one mother from Group III who was 20 years old. a Difference with Group I was statistically significant, P < 0.05. Comparisons were made adjusting to 10,000 cells. Table 7 Percentages of hyperdiploid cells identified by FISH Group
n
Total spreads
Spreads with trisomy 21 (%)
Total hyperdiploid spreads
Total genome hyperdiploid (%)
I II III
10 44 6
10321 44009 5875
8 (0.08) 96 (0.21)a 45 (0.76)a,b
8 (0.08) 98 (0.2)a,c 45 (0.76)a,b
0.9d 2.5d 8.8d
Difference with Group I was statistically significant, P < 0.05. Difference with Group II was statistically significant, P < 0.05. c Includes trisomy 13 cells. No trisomy 13 cells were found in Groups I and III. d The percentage of total genome hyperdiploids was estimated adding the number of chromosome 13 and 21 hyperdiploids (total hyperdiploid spreads) and multiplying by 23/2. a
b
and 8.8% for Groups I–III, respectively, and differences among groups remained significant. However, these estimates were 5-, 5-, and 9-fold higher than the total hyperdiploidy percentages directly observed by GTG-banding (Tables 5–7). 3.4. Mosaicism demonstrated by both classical and molecular cytogenetic techniques Only individuals with more than two trisomic cells by classical cytogenetic analysis and with trisomic
cell percentages >0.3%, according to Anastasi’s criteria were considered as true mosaics. More than 0.3% trisomic cells were not identified in any of the Group I parents. The same four individuals (two from Group II and two from Group III) previously identified as mosaic by classical cytogenetics were confirmed as true mosaics by FISH analysis (Table 8). The frequency of mosaicism was significantly higher among the recurrent DS parents as compared to that in parents with only one DS child (P < 0.01).
Table 8 Trisomy 21 mosaicism in parents of DS children Group
Family code/father or mother
II
P4/Moa
III
Individual percentage of mosaicism 47,X +21 GTG-banding
FISH
P19/Fa
1.75 1.5
0.5 0.5
P24/Fa P25/Fa
1.3 1.5
1 1.17
Group percentage of mosaicism
4.5 (2/44) 33.3 (2/6)
This table includes only Individuals considered as true mosaics, i.e. positive by both classical and molecular cytogenetic studies. No Group I parents were mosaic. a In addition to trisomy 21, this mother showed two cells with the robertsonian translocation 45,XX,der (13;14)(q10;q10), and three cells with other numerical abnormalities.
S. Frias et al. / Mutation Research 520 (2002) 25–37
4. Discussion 4.1. Population of study We investigated parental trisomy 21 mosaicism and the frequency of trisomic cells in 30 couples aged ≤35 years, 25 of which had one or more FT21 offspring. A high number of metaphases were analyzed to be able to identify cryptic mosaicism (1–2%) with a 95% confidence level according to Hook’s tables [32]. According to the pedigree analysis (not shown), the mean number of pregnancies was 1.8, 2.1 and 4.0 in Groups I–III, respectively, and there was no evidence of reduced fecundity in Groups II and III (Tables 1–3). An excess of male FT21 offspring was observed as the male/female sex ratio was 2.1. There is a well-known sex ratio deviation in FT21 individuals, estimated between 1.1 and 1.7 as compared to 1.06 in the general population [6,7,11,37,38]. Our data confirm this sex ratio deviation. The higher difference observed here may be attributed to chance alone. 4.2. Parental mosaicism of trisomy 21 Trisomy 21 mosaicism was found in 4/50 (8%) individuals with FT21 offspring. Among recurrent FT21 couples, trisomy 21 mosaicism was identified in 2/6 individuals (33.3%). This was expected, as parental mosaicism is one of the established causal factors of recurrent DS. Several isolated reports have documented parental mosaicism as the cause of recurrent FT21 [13–29]. Moreover, studies designed to identify parental mosaicism in recurrent FT21 families report high percentages of affected parents. In 1971, Hsu et al. [22] studied three recurrent FT21 families, finding all three fathers (3/6 individuals; 50%) to be trisomy 21 mosaics; Pangalos et al. [30] found parental mosaicism in 5/13 phenotypically normal couples with recurrent FT21, that is 5/26 (19%) parents (one father and four mothers). In 1998, James et al. [39] found trisomy 21 mosaicism in 2/4 recurrent FT21 families, the affected parent being the mother (aged <35 years) in both cases. Parental mosaicism has been found to be the main cause of sibling recurrence FT21, and our data agree with this observation. Recurrent FT21 may occur in families without cytogenetically diagnosed parental mosaicism when the
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trisomic cell line is confined to the germ cells or because of other etiological factors. The following causal factors other than parental mosaicism have been proposed [40–42]: (a) parental consanguinity-related genetic predisposition favoring non-disjunction; (b) chromosomal structural rearrangements distorting the meiotic process; (c) environmental predisposing factors; and (d) chance alone. No cases of parental consanguinity or chromosomal structural rearrangements were identified in our study. We observed parental trisomy 21 mosaicism in 2/44 (4.5%) individuals with only one FT21 product (Group II). Harris et al. [42] reviewed five cytogenetic surveys including a total of 200 couples with a single FT21 product; only 20–50 spreads were analyzed in most parents. Overall, 1.6% parents were identified as trisomy 21 mosaics, which is lower than the percentage reported here (4.5%). However, the analysis of 50 cells can only identify mosaics with ≥6–9% abnormal cells [32]. Our results and those previously reported in the medical literature suggest that parental mosaicism could be present in at least 3% of young couples with FT21 offspring, and when intentionally sought for, may be found as an important cause of FT21 (recurrent or isolated) in couples <35 years old [42,43]. Moreover, parental mosaicism may be part of the 5% of the paternal origin FT21 cases [6]. It is important to point out that in clinical practice, finding two cases of parental mosaicism among 44 parents of one FT21 product does not justify practicing the classical and molecular cytogenetic analyses described here in order to identify couples at high risk of conceiving a second DS product. This finding does, however, stress the need for prenatal diagnosis in all future pregnancies once a FT21 case has occurred. 4.3. Comparison of aneuploidy frequencies obtained by GTG-banding and FISH analyses The trisomy 21 frequencies obtained by GTGbanding were very similar to those obtained by FISH: 0.05 and 0.08 for Group I; 0.21 and 0.21 for Group II; 0.56 and 0.76 for Group III. Trisomy 13 cells were found only in individuals from Group II, with frequencies estimated at 0.007 by GTG-banding and 0.003 by FISH. Although trisomy 21 frequencies observed by both methods were very similar, the frequency of trisomy 13 according to FISH analysis was a half
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of that observed on GTG-banded metaphase, but still within the range of frequencies of hyperdiploidy reported for autosomes [44]. We found that the total genome hyperdiploidy percentage was significantly higher (P < 0.05) when assessed by FISH than when assessed by GTG-banding (Tables 5 and 7). It was assumed that the frequency of non-disjunction is similar for all chromosomes to estimate this percentage, and this criterion is applied in most cases extrapolating data from one or a few chromosomes to the entire genome. However, according to previously reported data [45–49] and to our results, non-disjunction frequencies are not similar for all chromosomes (Table 5). Indeed, the highest trisomy frequencies involved chromosomes 21 and X, while we failed to identify trisomies involving chromosomes 1–8, 11, 12, 16, 18, 19 and Y among a total of 13,809 GTG banded cells. Thus, estimating total genome hyperdiploidy percentages based on data from only one or a few chromosomes is not useful to estimate the actual total genome trisomy frequency.
hyperdiploid cells (0.004–0.18% per chromosome) using conventional cytogenetic techniques. Considering 21, non-21 and marker chromosome trisomies, numerical chromosome abnormality frequencies were higher among the parents of one or more FT21 offspring (Groups II and III) as compared to the reference Group I (P < 0.05). Marker chromosomes were always supernumerary, implying the occurrence of at least two events: one altering the chromosome structure, and the other causing non-disjunction. This finding is in accordance with the report of Juberg et al. [10] who studied the frequency of hyperdiploidy in 21,841 cells of 717 individuals attending a cytogenetic diagnosis laboratory over a 5.4-year period. The authors found 15.8 hyperdiploid cells/2000 cells analyzed (0.79%) among parents of aneuploid offspring (the chromosomes involved were not specified), but only 2.56 hyperdiploid cells/2000 cells analyzed (0.13%) among the remainder individuals studied. The results of this and Juberg’s studies suggest that the frequency of aneuploidy is higher in parents of aneuploid offspring.
4.4. Frequency of hyperdiploid cells
4.5. Possible non-disjunction predisposing factors
Classical cytogenetic techniques allowed the observation of hyperdiploid cells involving trisomies for several non-21 chromosomes (Tables 5 and 6). The second most frequent trisomy involved the X chromosome (Table 5). It has been suggested that the X chromosome is more susceptible to mal-segregation than autosomes [50,51]. X chromosome aneuploidy has been frequently reported in mothers of DS children affecting 1.2–2% of the analyzed cells, and has been considered as a risk factor for DS offspring [10,29]. Several studies have proposed that non-disjunction involves chromosomes 21 and X more frequently than other chromosomes [10,47,50,51]. Our data are in agreement with these observations. This may occur because of positive selection of cells bearing these trisomies or higher mal-segregation frequencies for chromosomes X and 21. The hyperdiploidy frequencies found in couples with normal descendants (Group I; Tables 5 and 6) are similar to the frequencies previously reported by Eastmond and Pinkel [44] who found 0.5/1000 hyperdiploid cells using FISH probes for chromosomes 9 and X and Cimino et al. [45] who found 0.2/1000
Non-disjunction predisposing factors can be intrinsic or extrinsic [7,11,29,41,52,53]. Intrinsic factors such as constitutional heterochromatic polymorphisms [39], consanguinity, abnormal folate metabolism, genetic predisposition to non-disjunction and parental mosaicism, have been previously proposed [7,8,41,52]. In this study, aneuploidy frequencies were similar in individuals with and without polymorphisms, ruling out an interchromosomal effect. Moreover, all couples included in the study denied consanguinity, making the presence of an autosomal recessive gene responsible for predisposition to non-disjunction unlikely. Recombination frequency and advanced maternal age have been conclusively associated to FT21 [11,30]. We did in fact find evidence of an age effect on the frequency of aneuploidy, as previously reported [46–48]. Although all individuals were ≤35 years old and the mean age was very similar in all groups, the frequency of trisomy 21 and of other hyperdiploid cells was significantly higher in Group IIb (parents aged 25–35 years) than in Group IIa (parents aged 15–24 years; P < 0.05). However, on comparing
S. Frias et al. / Mutation Research 520 (2002) 25–37
Groups I and IIb, with no age differences (Table 6), hyperdiploid cell frequencies remained significantly higher in Group IIb. This suggests that although the effect of age is important, it is not the only factor involved. Extrinsic factor causing non-disjunction may be related to personal habits or environmental exposure. It is noteworthy that 20/26 Group II parents with hyperdiploidy were couples (both parents had aneuploidies in 10/13 couples), and 12/18 parents without hyperdiploidy were couples. Because none of these couples were consanguineous, this high concordance (16/22 concordant couples = 73%, binomial test P < 0.05) may be indicative of the role of extrinsic factors in the generation of aneuplidy. Personal habits including smoking, alcohol consumption, drug consumption or exposure to environmental factors such as radiation, have been proposed as possible causal factors of aneuploidy [52]. Because this study was not designed to determine causal factors, data are insufficient to draw any conclusions. However, it seems that factors such as the father’s profession or living in Mexico City metropolitan zone are unrelated to these events as all couples from Group I, and 20/25 couples with FT21 children lived in Mexico City. Only two couples from Group I admitted being smokers, and only one hyperdiploid cell was found in these smoking parents, while no hyperdiploid cells were observed in the only Group II couple who admitted smoking. Cigarette smoking has been considered as a potential etiological factor for DS [52]. Yang et al. [54] found a significant association between maternal smoking and FT21 with the effect confined to young mothers and MII cases. Other studies have failed to find this association or have even reported a negative association between smoking mothers and DS offspring. This be related with higher susceptibility of the DS product to the components of cigarette smoke, increasing the frequency of abortion of DS fetuses and, thus, diminishing the frequency of live DS births to smoking mothers [55–57]. However, it is known that cigarette smoke contains aneugenic agents and increases non-disjunction frequencies of specific chromosomes such as 13 and Y in sperm [57,58]. Specific studies should be designed to further investigate this candidate factor.
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Two of the DS mothers were asthmatic. This factor deserves further study, as certain bronchodilator medications such as theophylline are known to cause chromosomal aberrations. One of these asthmatic mothers (P4) had a high frequency of hyperdiploid metaphases and structural chromosomal aberrations (Tables 5 and 8); her clinical background included two fetal losses and one FT21 child. These data may be related with constant exposure to genotoxic environmental compounds and/or medications. Even though the sample size is very small, in Group II predisposition to non-disjunction or extrinsic factors may lead to a higher risk of hyperdiploid cells and DS offspring. Furthermore, these couples may also have a higher risk of unborn products affected with other non-21 trisomies. The high frequency of miscarriages among our FT21 couples (17 and 25% for Groups II and III, respectively) supports the latter statement. In clinical practice, the identification of a 21 trisomy cell line is of uttermost importance for appropriate genetic counseling; parental 21 trisomy mosaicism identified in peripheral lymphocytes makes prenatal diagnosis mandatory for future pregnancies. The precise recurrence risk for a mosaic individual depends on the percentage of trisomic cells in the gonads. Karyotype analyses, however, are mostly performed on somatic cells because of the difficulty in analyzing gonadal tissue [29,59,60]. Currently, aneuploidy can be identified in sperm by FISH analysis, and the technique will be available for clinic diagnosis in a near future [61,62]. The present study sought to analyze possible causal factors for FT21 cases born to young couples, as the majority of DS children are born to young parents. Up to date the relevance of parental mosaicism in the origin of FT21 has not been clearly established. Recognized etiological factors such as maternal age and recombination abnormalities do not explain the origin of most FT21 cases, so that several other factors must be causally involved. The role of extrinsic factors such as personal habits and environmental exposure remain to be determined. Acknowledgements The authors wish to thank Psic. Gerardo Barragán for his help with the statistical analysis and Dr. Ma. Teresa Villarreal for reviewing the language.
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References [1] F. Salamanca (Ed.), Citogenética Humana, 1a Edición, Editorial Médica Panamericana, Mexico, 1990, pp. 117–128. [2] B. Dagna, M. Pierluigi, M. Grasso, P. Strigini, Origin of extra chromosome 21 in 343 families: cytogenetic and molecular approaches, Am. J. Med. Suppl. 7 (1990) 129–132. [3] M. Nadal, S. Moreno, M. Pritchard, M.A. Preciado, DS: characterization of a case with partial trisomy of chromosome 21 owing to a paternal balanced translocation (15;21)(q26;q22.1) by FISH, J. Med. Genet. 34 (1997) 50–54. [4] F. Ballesta, R. Queralt, D. Gómez, E. Solsona, Parental origin and meiotic stage of non-disjunction in 139 cases of trisomy 21, Ann. Genet. 42 (1999) 11–15. [5] R. McKinlay, G. Sutherland (Eds.), Chromosome abnormalities and Genetic counseling, DS: Other Full Aneuploidies, and Polyploidy, Oxford University Press, New York, 1996, pp. 244–335. [6] M.B. Petersen, S.E. Antonarakis, T.J. Hassold, S.B. Freeman, S.L. Sherman, D. Avramopoulos, M. Mikkelsen, Paternal nondisjunction in trisomy 21: excess of male patients, Hum. Mol. Genet. 2 (1993) 1691–1695. [7] A. Savage, M. Petersen, D. Pettay, I. Taft, Elucidating the mechanisms of paternal non-disjinction of chromosome 21 in humans, Hum. Mol. Genet. 7 (1998) 1221–1227. [8] T. Hassold, M. Abruzzo, A. Kenneth, D. Griffin, Human aneuploidy: incidence, origin and etiology, Environ. Mol. Mutagen. 28 (1996) 167–175. [9] C.A. Huether, J. Ivanovich, B.S. Goodwin, E.L. Krivchenia, Maternal age specific risk rate estimates for DS among live births in whites and other races from Ohio and Metropolitan Atlanta, 1970–1989, J. Med. Genet. 35 (1998) 482–490. [10] R. Juberg, J. Knops, P. Mowrey, Increased frequency of lymphocytic mitotic non-disjunction in recurrent spontaneous aborters, J. Med. Genet. 22 (1985) 32–35. [11] M.B. Petersen, M. Mikkelsen, Nondisjunction in trisomy 21: origin and mechanisms, Cytogenet. Cell Genet. 91 (2000) 199–203. [12] S. Armendares, L. Buentello, F. Salamanca, Frecuencia de mixoploid´ıas en 85 casos ´ındice con s´ındrome de Down, Rev. Inv. Clin. 42 (1990) 103–107. [13] C.E. Blank, E. Gemmel, M.D. Casey, M. Lord, Mosaicism in a mother with a mongol child, Br. Med. J. 2 (1962) 378–380. [14] D.W. Smith, E.M. Therman, K.A. Patau, S.L. Inhorn, Mosaicism in mother with two mongols, Am. J. Dis. Child. 104 (1962) 534. [15] E.D. Weinstein, J. Warkany, Maternal mosaicism and Down’s syndrome (mongolism), J. Pediatr. 63 (1963) 599–604. [16] S. Ferier, Enfant mongolien-parent mosaique: étude de deux families, J. Genet. Hum. 13 (1964) 315–336. [17] H. Verresen, H. Van den Berghe, J. Creemers, Mosaic trisomy in the phenotypically normal mother of a mongol, Lancet 1 (1964) 526–527. [18] D. Aarskog, Down’s syndrome transmitted trough maternal mosaicism, Acta Paediatr. Scand. 58 (1969) 609–614. [19] M. Mikkelsen, A Danish survey of patients with Down’s syndrome born to young mothers, Ann. N. Y. Acad. Sci. 171 (1970) 370–378.
[20] E. Krmpotic, M.B. Hardin, Secondary nondisjunction causing regular trisomy 21 in the offspring of a mosaic trisomy 21 mother, Am. J. Obstet. Gynecol. 110 (1971) 589–590. [21] J. Timson, R. Harris, R.L. Gadd, M.E. Ferguson-Smith, Down’s syndrome due to maternal mosaicism and the value of antenatal diagnosis, Lancet 1 (1971) 549–550. [22] L. Hsu, M. Gertner, E. Leiter, K. Hirschhorn, Paternal trisomy 21 mosaicism and Down syndrome, Am. J. Hum. Genet. 23 (1971) 592–601. [23] K. Mehes, Paternal trisomy 21 mosaicism and Down’s anomaly, Humangenetik 17 (1973) 297–300. [24] S. Kaffe, L.Y.E. Hsu, K. Hirschhorn, Trisomy 21 mosaicism in a young woman with two children with trisomy 21 Down’ syndrome, J. Med. Genet. 11 (1974) 378–379. [25] Z. Papp, K. Cséscei, J. Skapinyerz, B. Dolhay, Paternal normal-trisomy 21 mosaicism as an indication for amniocentesis, Clin. Genet. 6 (1974) 192–194. [26] B.W. Richards, Investigation of 142 mosaic mongols and mosaic parents of mongols: cytogenetic analysis and maternal age at birth, J. Med. Def. Res. 18 (1974) 199–208. [27] F. Nuzzo, M. Stefanini, G. Simoni, A family with a three sibs carrying trisomy 21 mosaicism, Ann. Genet. 18 (1975) 111–116. [28] A. Osuna, A. Moreno, Regular G21-trisomy in 3 sibs from mother with trisomy 21 mosaicism, J. Med. Genet. 14 (1977) 286–287. [29] D.S. Krishna-Murthy, T.I. Farag, Recurrent regular trisomy 21 in two Bedouin families. Parental mosaicism versus genetic predisposition, Ann. Genet. 38 (1995) 217–224. [30] C. Pangalos, C.C. Talbot Jr., J.G. Lewis, P.A. Adeisberger, DNA polymorphism analysis in families with recurrence of free trisomy 21, Am. J. Hum. Genet. 51 (1992) 1027–1051. [31] J. Moorehead, P. Nowel, W. Mellman, D. Battips, Chromosome preparations of leucocytes cultured from human principal blood, Exp. Cell. Res. 20 (1960) 613–616. [32] E.B. Hook, Exclusion of chromosomal mosaicism: tables of 90, 95 and 99% confidence limits and comments on use, Am. J. Hum. Genet. 29 (1977) 94–97. [33] R. Lisker, Hallazgos citogenéticos en padecimientos hematológicos malignos, Rev. Invest. Clin. (Mex.) 39 (1987) 187–196. [34] S.L. Gersen, M.B. Keagle, Principles of clinical cytogenetics. Humana Press, NJ, 1999, pp. 73–76. [35] J. Anastasi, M. Le Beau, J. Vardiman, C. Westbrook, Detection of numerical chromosomal abnormalities in neoplastic hematopoietic cells by in situ hybridization with a chromosome-specific probe, Am. J. Pathol. 136 (1990) 131– 139. [36] A. Elhajouji, F. Tibaldi, M. Kirsch-Volders, Indication for a thresholds of chromosome non-disjuntion versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes, Mutagenesis 12 (1997) 133–140. [37] O.M. Mutchinik, R. Lisker, V. Babinski, Programa mexicano de registro y vigilancia epidemiológica de malformaciones congénitas externas, Salud Publica Méx. 30 (1988) 88–100. [38] D.K. Griffin, M.A. Abruzzo, E.A. Millie, E. Feingold, T.J. Hassold, Sex ratio in normal and disomic sperm: evidence that
S. Frias et al. / Mutation Research 520 (2002) 25–37
[39]
[40]
[41]
[42]
[43]
[44]
[45] [46]
[47]
[48]
[49]
[50]
the extra chromosome 21 preferentially segregates with the Y chromosome, Am. J. Hum. Genet. 59 (1996) 1108–1113. R.S. James, K. Ellis, D. Pettay, P.A. Jacobs, Cytogenetic and molecular study of four couples with multiple trisomy 21 pregnancies, Eur. J. Hum. Genet. 6 (1998) 207–212. S.K. Murthy, K. Prabhakara, Mitotic disturbance associated with inversion 9qh: a case report, Ann. Genet. 33 (1990) 169–172. O. Alfi, R. Chang, S. Azen, Evidence for genetic control of nondisjunction in man, Am. J. Hum. Genet. 32 (1980) 477– 483. D. Harris, M. Begleiter, J. Chamberlin, L. Hankis, Parental trisomy 21 mosaicism, Am. J. Hum. Genet. 34 (1982) 125– 133. E.S. Sachs, M.G.J. Jahoda, F.J. Los, L. Pijpers, J.W. Wladimiroff, Trisomy 21 mosaicism in gonads with unexpectedly high recurrence risks, Am. J. Med. Genet. 7 (1990) 186–188. D.A. Eastmond, D. Pinkel, Detection of aneuploidy and aneuploidy-inducing agents in human lymphocytes using fluorescence in situ hybridization with chromosome-specific DNA probes, Mutat. Res. 234 (1990) 303–318. M.C. Cimino, R.R. Tice, J.C. Liang, Aneuploidy in mammalian cells in vivo, Mutat. Res. 167 (1986) 107–122. P.A. Jacobs, W.M.C. Brown, Distribution of human chromosome counts in relation to age, Nature 191 (1961) 1178–1180. S.M. Galloway, K.E. Buckton, Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding, Cytogenet. Cell Genet. 20 (1978) 78–95. M.A. Bender, R.J. Preston, R.C. Leonard, B.E. Pyatt, P.C. Gooch, M.D. Shelby, Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample, Mutat. Res. 204 (1988) 421–433. L. Vega, M.E. Gonsebatt, P. Ostrosky-Wegman, Aneugenic effect of sodium arsenite on human lymphocytes in vitro: and individual susceptibility effect detected, Mutat. Res. 334 (1995) 365–373. A. Carere, A. Antoccia, D. Cimini, R. Crebelli, F. Degrassi, P. Leopardi, F. Marcon, A. Sgura, C. Tanzarella, A. Zijno, Análisis of chromosome loss and non-disjunction in cytokinesis-blocked lymphocytes of 24 male subjects, Mutagenesis 14 (1999) 491–496.
37
[51] J. Catalán, J. Surrallés, G.C.M. Falck, K. Autio, H. Norppa, Segregation of sex chromosomes in human lymphocytes, Mutagenesis 15 (2000) 251–255. [52] T. Hassold, P. Hunt, To err (meiotically) is human: the genesis of human aneuploidy, Nat. Rev. Genet. 2 (2001) 280– 291. [53] S. Antonarakis, S. Kittur, C. Metaxotou, P. Watkins, Analysis of DNA haplotypes suggests a genetic predisposition to trisomy 21 associated with DNA sequences on chrosome 21, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 3360–3364. [54] Q. Yang, S.L. Sherman, T.J. Hassold, K. Allran, L. Taft, D. Pettay, M.J. Khoury, J.D. Erickson, S.B. Freeman, Risk factors for trisomy 21: maternal cigarette smoking and oral contraceptive use in a population-based case–control study, Genet. Med. 1 (1999) 80–88. [55] C. Chen, T.J. Gilbert, J.R. Daling, Maternal smoking and Down syndrome: the confounding effect of maternal age, Am. J. Epidemiol. 149 (1999) 442–446. [56] E.B. Hook, P.K. Cross, Maternal cigarette smoking, Down syndrome in live births, and infant race, Am. J. Hum. Genet. 42 (1988) 482–489. [57] Q. Shi, E. Ko, L. Barclay, T. Hoang, A. Rademaker, R. Martin, Cigarette smoking and aneuploidy in human sperm, Mol. Reprod. Dev. 59 (2001) 417–421. [58] J. Rubes, X. Lowe, D. Moore II, S. Perreault, V. Slott, D. Evenson, S.G. Selevan, A.J. Wyrobek, Smoking cigarettes is associated with increased sperm disomy in teenage men, Fertil. Steril. 70 (1998) 715–723. [59] F. Vogel, A.G. Motulsky, Human genetics: problems and approaches, Mutation, Spnnger, Berlin, 1974, pp. 291–292, Chapter 5. [60] I. Uchida, V. Freeman, Trisomy 21 Down syndrome. II. Structural chromosome rearrangements in the parents, Hum. Genet. 72 (1986) 118–122. [61] A. Wyrobek, X.R. Lowe, Multiprobe fluorescence in situ hybridization methods for detecting chromosomally defective sperm in mice and humans, in: M. Andreeff, D. Pinkel (Eds.), Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications, Wiley, New York, 1999, pp. 391–408. [62] S. Frias, P. Van Hummelen, M. Meistrich, X. Lowe, M.D. Shelby, A.J. Wyrobek, NOVP chemotherapy induces transient increases in disomy 18 and 21 in sperm of Hodgkin’s disease patients, Am. J. Hum. Genet. S 63 (1998) 758.
Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring Sara Frias a,∗ , Sandra Ramos a , Bertha Molina a , Victoria del Castillo a , Dora Gilda Mayén b a
Laboratorio de Citogenética, Departamento de lnvestigación en Genética Humana, Instituto Nacional de Pediatr´ıa, Insurgentes Sur 3700-C, Col. Insurgentes-Cuicuilco, Mexico DF, CP 04530, Mexico b Servicio de Genética, Instituto Nacional de Perinatolog´ıa, Mexico DF, CP 04530, Mexico Received 10 January 2002; received in revised form 19 June 2002; accepted 19 June 2002
Abstract Down syndrome (DS) resulting from free trisomy 21 (FT21) has been largely associated with advanced maternal age. However, approximately 60% of FT21 cases are born to young couples. Thus, the etiological factors responsible for these FT21 children must differ from those proposed for maternal age-related FT21. These factors have not been defined. In this study, we analyzed the chromosomes of peripheral blood lymphocytes from three groups of couples aged ≤35 years, to identify chromosomal trisomies: Group I included 5 couples with normal offspring; Group II included 22 couples with one FT21 child; and Group III consisted of 3 couples with recurrent FT21. A total of 13,809 metaphases were analyzed with G-banding and 60,205 metaphases were analyzed with FISH using a 13/21 centromeric probe. Aneuploidy was significantly more frequent in Groups II and III. The frequencies of hyperdiploid cells were 0.19, 0.49 and 0.96% in Groups I–III, respectively. FISH analysis showed that trisomy 21 cell percentages were 0.08, 0.21 and 0.76 for Groups I–III, respectively, and were very similar to those obtained with G-banding. Trisomy 21 mosaicism was found in 2/22 couples with one FT21 offspring, and in 2/3 couples with recurrent FT21. Our data suggest that mosaicism is an important cause of FT21 offspring in young couples, and that aneuploidy is more frequent among couples with FT21 offspring. This may be related with age and other undetermined intrinsic and extrinsic factors. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Trisomy 21; Parental mosaicism; Aneuploidy; Lymphocytes; Down syndrome; Etiology of trisomy 21
1. Introduction Down syndrome (DS) is the most frequent chromosomal disorder in humans, occurring in 1 of every 700 live births [1]. DS has five cytogenetic variants: (a) free trisomy 21 (FT21), defined as the presence of an extra chromosome 21 (FT21); (b) translocations involving a rearrangement of one chromosome 21 with ∗ Corresponding author. Tel.: +82-5255-5606-9580; fax: +82-5255-5606-9455. E-mail address: [email protected] (S. Frias).
a D- or a G-Group chromosome; (c) 21q21q isochromosome; (d) cellular mosaicism, defined as the presence of two different cell lines in the same individual, generally one normal and one trisomic line in variable proportions; and (e) partial trisomy of the 21q22.3 region [2–4]. In 95% of FT21 cases, the additional chromosome 21 is of maternal origin, and the remainder 5% is of paternal origin. Two of every 10 maternal origin cases and 3 of every 4 paternal origin cases, originate from non-disjunction during meiosis II [2–7]. While maternal origin FT21 is clearly correlated with advanced
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age [1,8,9], paternal origin FT21 is not age-related [7]. However, although women above age 35 years have a higher risk of having FT21 offspring, <5% of all FT21 births occur to these women. Approximately 60% of FT21 cases are born to young couples [9,10]. Thus, the etiological factors of the latter and paternal origin FT21 must differ from those proposed for maternal age-related FT21 (for review, see [6,11]), and have not been clearly defined. Parental mosaicism is one of the main causes of recurrent FT21, and is also a cause of FT21 in young couples. Mosaicism is not exclusive to DS patients, as two different cell lines may be present in phenotypically normal individuals with a low frequency of trisomic cells. The abnormal cell line may be confined to one or more specific tissues, such as germ or blood cells, with no phenotypic consequences. This anomaly may remain unnoticed even after routine cytogenetic diagnosis, when the number of cells analyzed is insufficient to identify abnormal lines present in <10% of cells, and because of the preferential proliferation of normal as compared to trisomic lines [12]. In addition, if one of the parents has cryptic trisomy 21 mosaicism in the germ cells, he or she could be phenotypically normal, have a normal karyotype in lymphocytes, but have an increased risk of having FT21 offspring. There are many reports of recurrent FT21 infants born to mosaic parents [13–29]. Parental mosaicism as a cause of FT21 has been examined in few studies, with only 20–200 cells being analyzed [22,30]. We, therefore, investigated the presence of trisomy 21 mosaicism in parents of FT21 offspring performing chromosome analysis on a large number of peripheral blood lymphocyte metaphases by classical GTG-banding and fluorescence in situ hybridization (FISH). This enabled us to detect low levels of parental mosaicism as part of the etiology of FT21 in young couples.
2. Materials and methods 2.1. Subjects The study population consisted of Mexican couples with one or more FT21 offspring. All couples attended the Genetics Outpatient Services of the National
Institute of Pediatrics and the National Institute of Perinatology of Mexico from 1997 to 1999. To avoid advanced maternal age-related bias, we included only couples whose ages were ≤35 years at the time their FT21 offspring were born. Five volunteer Mexican couples aged ≤35 years with healthy offspring were included as a control group. All individuals were asked to complete a questionnaire that included family history and pedigree. Couples with a history of previous chemotherapy and/or radiotherapy or who had a viral infection within 1 week before sampling were excluded. A code was assigned to each couple. Parents of healthy offspring were coded as C1–C5 followed by Fa for ‘father’ or Mo for ‘mother’. Parents of FT21 offspring were coded as P1–P25; P22–P25 were recurrent FT21 cases. 2.2. Cell cultures and cytogenetic procedures Heparinized whole blood samples (0.5 ml) were drawn from all subjects, and cultured in 5 ml modified McCoy 5a medium (Gibco, USA) containing 0.25 ml phytohemagglutinin (Gibco, USA) and 0.1 ml reconstituted penicillin–streptomycin (Gibco, USA). Cultures were incubated at 37 ◦ C and harvested after 72 h using 0.1 ug/ml of colcemid (Gibco, USA) for 1 h, according to the standard method described by Moorehead et al. [31]. Cells were dropped on cold slides and air-dried to be processed for both classical and molecular cytogenetic studies. For GTG-banding, slides were treated with 0.015% trypsin (Gibco, USA) and 0.01% EDTA (Sigma, USA) in phosphate buffer (pH7). Chromosomes were stained with 5% Giemsa (Gibco, USA) in phosphate buffer. All slides were coded by an independent individual to ensure blind scoring. FISH was performed using a digoxigenin-labeled centrometric probe for chromosomes 13/21 (D13Z1/ D21Z1, Oncor kit, Gaithersburg, MD, USA). Chromosomes were denatured with 70% formamide with 2XSSC at 72 ◦ C for 2 min, and hybridized at 37 ◦ C for 24 h. Slides were washed with 0.2XSSC solution at 72 ◦ C for 2–5 min; chromosomes were incubated with fluorescein-labeled anti-digoxigenin antibodies (Oncor kit, Gaithersburg, MD, USA) for 30 min at 37 ◦ C and washed three times with 0.5% Tween 20 and 4XSSC for 2 min. Chromosomes were
S. Frias et al. / Mutation Research 520 (2002) 25–37
then counterstained with propidium iodine (Oncor kit, Gaithersburg, MD, USA). 2.3. Chromosome analysis The classical cytogenetic analyses included: (a) karyotype analysis of 15 GTG-banded metaphases per individual, examining the number and structure of all chromosome regions and subregions to identify constitutional aberrations; and (b) analysis of 200–400 GTG-banded non-adjacent metaphases to identify numerical chromosomal abnormalities [32]. In one couple (P2) only 190 and 165 metaphases per individual could be analyzed due to technical problems with the lymphocyte cultures. Whenever an aneuploid metaphase was identified, the chromosome number and chromatin condensation of all metaphases within a radius equivalent to the 10× objective lens field were also analyzed, in order to rule out false gain of chromosomes caused by technical artifacts. Technical artifact-related aneuploid metaphases were not considered for the analysis. For FISH analysis, metaphases were examined on an Olympus BX40 fluorescence microscope using triple band filters for DAPI, FITC and Texas red, and single filters for FITC and Texas red. At least 1000 metaphases per individual were scored in 57 parents, while only 965, 957 and 852 cells were analyzed in the three remainder. Fluorescent signals observed exactly on chromosome centromeres of similar size and intensity were considered as positive. Metaphases showing four signals (two on G chromosomes and two on D chromosomes) were considered as normal. Metaphases were considered as trisomic for chromosome 21 when three signals were observed on G chromosomes and two signals were observed on D chromosomes. Because our purpose was to identify mosaicism for a trisomic cell line, only trisomies and not monosomies were included for the analysis. Furthermore, an undetermined percentage of monosomies may be caused by technical artifacts such as broken metaphases or lack of hybridization. 2.4. Definition of mosaicism An individual was defined as a true trisomy 21 mosaic only if: (a) classical cytogenetics showed more
27
than two cells exhibiting the same extra chromosome in the same sample [33,34] and (b) FISH analysis showed a frequency of trisomic cells greater than the mean percentage plus twice the standard deviation of the control group [35]. The power for detecting mosaicism according with the number of lymphocytes analyzed was: for a 95% confidence level when n = 200 cells are analyzed, the percent of mosaicism excluded is 2%; when n ≥ 400 cells are analyzed, the percent of mosaicism excluded is 1% [32]. Differences in the frequency of abnormal cells among the three groups were compared using the chi-square proportions test.
3. Results 3.1. Study population A total of 30 couples (60 individuals) aged ≤35 years were included: five with only normal offspring (Group I), and 25 with FT21 offspring. Twenty-two of these couples had only one FT21 child (Group II), and the remainder three couples had more than one FT21 child (recurrent DS, Group III). Background and family history of all participants are described in Tables 1–3. All couples belonging to Groups I and III, and 17/22 couples from Group II were Mexico City metropolitan zone (MMZ) residents. Mean parental age was 28.3, 26.2 and 25.7 years for Groups I–III, respectively. DS cases were the first child born to 11/22 couples in Group II and 2/3 couples in Group III. An excess of male DS cases was found, as the male/female sex ratio was 2.1 (19 males/9 females). 3.2. Classical cytogenetics Karyotypes were normal in all parents, as no constitutional (congenital) numerical or structural aberrations were found. However, constitutional chromosomal polymorphisms (normal variants) were observed in eight individuals (Table 4). Overall, 67 hyperdiploid cells were found among a total of 13,809 GTG banded cells (0.49%) distributed among all groups (Table 5). Supernumerary marker chromosomes (chromosomes resembling the
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S. Frias et al. / Mutation Research 520 (2002) 25–37
Table 1 Family background (Group I) Family code
Age (years)
Profession
Other data
Pregnancies/miscarriagesa
C1
Fa: 25 Mo: 20
Student Student
MMZ
1/0
C2
Fa: 30
MMZ
2/0
Mo: 30
Biologist working at beer factory Laboratory worker
Fa: 32
Administrative
MMZ, father smokes >5 cigarettes per day
2/0
Mo: 33
Laboratory worker
C4
Fa: 30 Mo: 29
Electronic engineer Computer engineer
MMZ
1/0
C5
Fa: 28 Mo: 26
Chauffer Housewife
MMZ Both parents smoke >5 cigarettes per day
3/0 (two induced abortions)
C3
Mean age: 28.3, S.D.: 38
Total pregnancies/miscarriages: 9/0
Age: at the time of birth of the last offspring; Fa: father; Mo: mother; MMZ: Mexico City metropolitan zone residents. a Only considered in miscarriages those spontaneous and not induced abortions. Table 2 Family background (Group II) Family code/sex of FT21 offspring
Age (years)
Profession
Other data
Pregnancies/miscarriagesa
P1/male
Fa: 18 Mo: 15
Merchant Housewife
MMZ
1/0
P2/male
Fa: 26
Builder
MMZ, chickenpox at week 13 of pregnancy
4/2
Mo: 27
Housewife
P3/male
Fa: 28 Mo: 21
Agronomy technician Housewife
MMZ
2/0
P4/female
Fa: 35
Lawyer
MMZ, asthmatic
5/3, one of the miscarriage products had a neural tube defect
Mo: 33
Housewife
P5/male
Fa: 23 Mo: 21
Carpenter Housewife
MMZ
2/0
P6/male
Fa: 25 Mo: 24
Office employee Housewife
MMZ
1/0
P7/male
Fa: 27 Mo: 26
Airline worker Housewife
Baja California Sur
2/0
P8/female
Fa: 34 Mo: 32
Copper plant worker Housewife
MMZ
1/0
P9/female
Fa: 35
Merchant
MMZ, father smokes 20 cigarettes per day, mother smokes 2 cigarettes per day
4/1
Mo: 34
Housewife
P10/male
Fa: 21 Mo: 21
Hardware employee Housewife
MMZ
1/0
S. Frias et al. / Mutation Research 520 (2002) 25–37
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Table 2 (Continued) Family code/sex of FT21 offspring
Age (years)
Profession
Other data
Pregnancies/miscarriagesa
P11/male
Fa: 27 Mo: 27
Not available Housewife
Chihuahua chih.
1/0
P12/male
Fa: 35 Mo: 33
Restaurant administrator Housewife
Veracruz ver.
2/0
P13/male
Fa: 28 Mo: 25
Grape cutter Housewife
Cuautla, mor.
3/0 (two induced abortions)
P14/female
Fa: 33 Mo: 30
Builder Housewife
MMZ
1/0
P15/male
Fa: 18 Mo: 16
Chauffer Housewife
MMZ
3/1
P16/male
Fa: 27 Mo: 26
Artisan (rug weaver) Housewife
Oaxaca oax.
3/1
P17/male
Fa: 20 Mo: 21
Waiter Housewife
MMZ
3/0
P18/male
Fa: 32 Mo: 30
Chauffeur Housewife
MMZ
3/0
P19/female
Fa: 33 Mo: 24
Data Non available
MMZ
1/0
P20/female
Fa: 27 Mo: 23
Carpenter Housewife
MMZ
1/0
P21/female
Fa: 16 Mo: 17
Storage employee Student
MMZ
2/0 (one induced abortion)
P22/male
Fa: 29 Mo: 27
Window cleaner Housewife
MMZ
1/0
Total male/ female: 15/7
Mean age: 26.2, S.D.: 5.62
Total pregnancies/miscarriages: 47/8 = 17% fetal lost
Age: at birth of the DS child; Fa: father; Mo: mother; MMZ: Mexico City metropolitan zone residents. a Only considered in miscarriages those spontaneous and not induced abortions. Table 3 Family background (Group III) Family code/sex of FT21 offspring
Age (years)
Profession
Relevant clinical data
Pregnancies/miscarriagesa
P23/two males
Fa: 25
Merchant
MMZ, asthmatic
3/1 (one elective abortion of a FT21 product)
Mo: 28
Housewife
Fa: 26
Builder
MMZ, one stillborn malformed child
4/0
Mo: 25
Housewife
P25/one male, one female
Fa: 25 Mo: 25
Grocery store employee Housewife
MMZ
5/2 (one stillborn)
Total male/female: 4/2
Mean age: 25.66, S.D.: 1.21
P24/one male, one female
Total pregnancies/miscarriages: 12/3 = 25% fetal lost
Age: at birth of the youngest DS offspring; Fa: father; Mo: mother; MMZ: Mexico City metropolitan zone residents. a Only considered in miscarriages those spontaneous and not induced abortions.
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S. Frias et al. / Mutation Research 520 (2002) 25–37
Table 4 Chromosomal polymorphisms in parents of normal and FT21 offspring Family code/father or mother
Karyotypea
I
C3/Fa
46,XY,22pstk−
II
P4/Mo P10/Mo P15/Fa P15/Mo P17/Fa P18/Mo P20/Fa
46,XX,inv(9) 46,XX,22pstk+ 46,XYqh+ 46,XX,21pstk+ 46,XY,inv(9) 46,XX,16qh+ 46,XYqh+
Group
No polymorphisms were observed among individuals belonging to Group III. a Notations: pstk−: decrease in length of the satellite stalk on the short arm; pstk+: increase in length of the satellite stalk on the short arm; qh+: increase in length of the heterochromatin on the long arm; inv(9): inversion of the chromosome 9.
morphology of a C–E or G chromosome, but not definitely identified) were found in 12 individuals (Table 5). Twelve of the 22 supernumerary markers were G-like, but did not involve chromosome 21 rearrangements as no extra 21 signal was observed by FISH in any case. The analysis of 200 or more GTG metaphases per individual revealed the presence of low percentages of trisomic cells in individuals from all three study groups. Trisomies involving normal chromosomes (non-markers) other than 21 were found in 11 individuals: 1 from Group I, 9 from Group II and 1 from Group III (Table 5). Only one trisomic cell involving chromosomes 10, 13, 17, 20, 22 or X was found in one individual from Group I, seven from Group II and one from Group III; while more than one trisomic cell was found in only two individuals: the asthmatic mother from Group II (P4/Mo) who had several aneuploid metaphases (two cells with trisomy 9, one cell with trisomy 17, and seven cells with trisomy 21); and one mother from Group II (P8/Mo) with three trisomic 47,XXX cells, representing a clone. A unique trisomic X cell was found in two more mothers; and trisomy X was the most frequent non-21 chromosome trisomy (Table 5). Chromosome 21 was more involved in mitotic non-disjunction than any other chromosome: 30/67 (44.7%) hyperdiploid cells had an extra chromosome 21. In Group I, 4/2200 (0.19%) metaphases were hyperdiploid, and trisomy 21 was found in only one.
In Group II, 51/10,355 (0.49%) metaphases were hyperdiploid, and 22 showed an additional 21 chromosome. In Group III, 12/1254 (0.96%) metaphases were hyperdiploid, and 7 had trisomy 21 (Tables 5 and 6). Overall, hyperdiploid metaphases and specifically trisomy 21 metaphases were significantly less frequent in Group I as compared to the groups with FT21 offspring (P < 0.05). Although mean age was similar in all groups, we assessed the effect of parental age by comparing the frequency of hyperdiploid metaphases in individuals aged 15–24 years and those aged 25–35 years. This sub-classification was feasible only in Group II, which included a sufficient number of subjects and showed the widest age dispersion (Table 1). Group IIa included 16 parents aged 15–24 years, and Group IIb included 28 parents aged 25–35 years (Table 6). The frequency of hyperdiploid and trisomy 21 cells was significantly higher in Group IIb than in Group IIa (P < 0.05) Moreover, even though the ages were similar the frequency of hyperdiploid cells was significantly higher (P < 0.05) in Group IIb than in Group I, mean age 28.3 years (only one mother aged <25 years). Although trisomy 21 cells were found in 17/60 individuals from all three groups, in most cases only one affected cell was identified. More than two trisomic 21 cells were found in 2/44 individuals belonging to Group II and in 2/6 individuals belonging to Group III (Table 5). 3.3. Molecular cytogenetics In accordance with the observations made by classical cytogenetics, FISH analysis revealed that chromosome 21 was significantly more involved in mitotic non-disjunction than chromosome 13. While only two individuals from Group II (P7/Mo and P18/Fa) showed one trisomy 13 cell, several individuals from all groups showed one trisomic 21 cell. Moreover, 11/44 subjects from Group II and 4/6 from Group III showed trisomy 21 frequencies above the threshold for mosaicism (0.3%). Percentages of trisomic 21 cells were 0.08, 0.21 and 0.76 for Groups I–III, respectively (Table 7). Tetraploid cells were identified in all groups, and the frequency of these cells was not significantly different among groups. FISH analysis revealed that total genome hyperdiploidy percentages calculated as described by Elhajouji [36] were 0.9, 2.5
S. Frias et al. / Mutation Research 520 (2002) 25–37
31
Table 5 Frequency of trisomic cells in parents of normal and FT21 offspring, with hyperdiploid cells Group Family code/ father or mother I
II
III
Total
#Metaphases Number (%) of trisomic cells for individual chromosomes 9
C2/Fa C3/Mo C5/Fa Total Group Ia
200 200 200 2200
P1/Mo P2/Mo P3/Fa P3/Mo P4/Fa P4/Mo P5/Fa P7/Fa P7/Mo P8/Fa P8/Mo P10/Fa P10/Mo P12/Fa P12/Mo P13/Fa P13/Mo P16/Fa P17/Fa P17/Mo P18/Fa P18/Mo P19/Fa P19/Mo P21/Mo P22/Mo Total Group IIa
200 165 400 400
P23/Fa P24/Fa P24/Mo P25/Fa P25/Mo Total Group IIIa
400 400 200 200 200 200 200 200 200 200 300 300 200 200 200 200 200 200 200 200 200 10355 200 230 224 200 200 1254 13809b
10
13
17
20
21
22
X
Markers
Total
1 (0.5) 1 (0.5)
1 2 1 4
(0.5) (1.0) (0.5) (0.19)
2 1 2 (0.5) 3 3 (0.75) 3 2 10 1 1 (0.5) 2 1 1 3 1 1 1 (0.5) 1 1 1 (0.3) 2 1 (0.3) 1 1 (0.5) 2 1 1 1 1 3 1 1 4 (2.0) 4 51
(1.0) (0.6) (0.75) (0.75) (0.5) (2.5) (0.25) (1.0) (0.5) (0.5) (1.5) (0.5) (0.5) (0.5) (0.5) (0.6) (0.3) (1.0) (0.5) (0.5) (0.5) (0.5) (1.5) (0.5) (0.5) (2.0) (0.49)
1 3 3 4 1 12
(0.5) (1.3) (1.3) (2.0) (0.5) (0.96)
1 (0.5) 1 (0.5)
2 (1.0)
1 (0.6) 1 (0.25)
2 (0.5)
2 7 1 1 1 1
1 (0.25)
(0.5) (1.75) (0.25) (0.5) (0.5) (0.5) 3 (1.5) 1 (0.5) 1 (0.5)
1 (0.5) 1 (0.3) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 3 (1.5) 1 (0.5) 1 (0.5)
1 (0.5) 3 (1.3) 3 (1.3) 1 (0.5)
3 (1.5) 1 (0.5)
2 1 1 2 (0.014) (0.007) (0.007) (0.014)
1 30 (0.007) (0.21)
3 5 22 (0.021) (0.036) (0.15)b
67 (0.49)
Only parents and chromosomes involved in hyperdiploidy are shown. a Total metaphases analyzed per group. b Total metaphases in 60 individuals from all groups. Aneuploidy frequencies for specific chromosomes were estimated based on this number.
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S. Frias et al. / Mutation Research 520 (2002) 25–37
Table 6 Comparison of hyperdiploid cell percentages observed with G-banding (according to age groups) Group
n
Age (years)
Total spreads
Cells with trisomy 21 (%)
Cells with non-21 trisomy (%)
Total hyperdiploids (%)
I IIa IIb II III
10 16 28 44 6
25–35 15–24 25–35 15–35 25–35
2200 4000 6355 10355 1254
1 2 20 22 7
3 10 19 29 5
4 12 39 51 12
(0.05) (0.05) (0.3)a (0.21)a (0.56)a
(0.14) (0.25) (0.3)a (0.28)a (0.40)a
(0.19) (0.30) (0.6)a (0.49)a (0.96)a
Groups I and III include 25–35-year-old individuals, except one mother from Group III who was 20 years old. a Difference with Group I was statistically significant, P < 0.05. Comparisons were made adjusting to 10,000 cells. Table 7 Percentages of hyperdiploid cells identified by FISH Group
n
Total spreads
Spreads with trisomy 21 (%)
Total hyperdiploid spreads
Total genome hyperdiploid (%)
I II III
10 44 6
10321 44009 5875
8 (0.08) 96 (0.21)a 45 (0.76)a,b
8 (0.08) 98 (0.2)a,c 45 (0.76)a,b
0.9d 2.5d 8.8d
Difference with Group I was statistically significant, P < 0.05. Difference with Group II was statistically significant, P < 0.05. c Includes trisomy 13 cells. No trisomy 13 cells were found in Groups I and III. d The percentage of total genome hyperdiploids was estimated adding the number of chromosome 13 and 21 hyperdiploids (total hyperdiploid spreads) and multiplying by 23/2. a
b
and 8.8% for Groups I–III, respectively, and differences among groups remained significant. However, these estimates were 5-, 5-, and 9-fold higher than the total hyperdiploidy percentages directly observed by GTG-banding (Tables 5–7). 3.4. Mosaicism demonstrated by both classical and molecular cytogenetic techniques Only individuals with more than two trisomic cells by classical cytogenetic analysis and with trisomic
cell percentages >0.3%, according to Anastasi’s criteria were considered as true mosaics. More than 0.3% trisomic cells were not identified in any of the Group I parents. The same four individuals (two from Group II and two from Group III) previously identified as mosaic by classical cytogenetics were confirmed as true mosaics by FISH analysis (Table 8). The frequency of mosaicism was significantly higher among the recurrent DS parents as compared to that in parents with only one DS child (P < 0.01).
Table 8 Trisomy 21 mosaicism in parents of DS children Group
Family code/father or mother
II
P4/Moa
III
Individual percentage of mosaicism 47,X +21 GTG-banding
FISH
P19/Fa
1.75 1.5
0.5 0.5
P24/Fa P25/Fa
1.3 1.5
1 1.17
Group percentage of mosaicism
4.5 (2/44) 33.3 (2/6)
This table includes only Individuals considered as true mosaics, i.e. positive by both classical and molecular cytogenetic studies. No Group I parents were mosaic. a In addition to trisomy 21, this mother showed two cells with the robertsonian translocation 45,XX,der (13;14)(q10;q10), and three cells with other numerical abnormalities.
S. Frias et al. / Mutation Research 520 (2002) 25–37
4. Discussion 4.1. Population of study We investigated parental trisomy 21 mosaicism and the frequency of trisomic cells in 30 couples aged ≤35 years, 25 of which had one or more FT21 offspring. A high number of metaphases were analyzed to be able to identify cryptic mosaicism (1–2%) with a 95% confidence level according to Hook’s tables [32]. According to the pedigree analysis (not shown), the mean number of pregnancies was 1.8, 2.1 and 4.0 in Groups I–III, respectively, and there was no evidence of reduced fecundity in Groups II and III (Tables 1–3). An excess of male FT21 offspring was observed as the male/female sex ratio was 2.1. There is a well-known sex ratio deviation in FT21 individuals, estimated between 1.1 and 1.7 as compared to 1.06 in the general population [6,7,11,37,38]. Our data confirm this sex ratio deviation. The higher difference observed here may be attributed to chance alone. 4.2. Parental mosaicism of trisomy 21 Trisomy 21 mosaicism was found in 4/50 (8%) individuals with FT21 offspring. Among recurrent FT21 couples, trisomy 21 mosaicism was identified in 2/6 individuals (33.3%). This was expected, as parental mosaicism is one of the established causal factors of recurrent DS. Several isolated reports have documented parental mosaicism as the cause of recurrent FT21 [13–29]. Moreover, studies designed to identify parental mosaicism in recurrent FT21 families report high percentages of affected parents. In 1971, Hsu et al. [22] studied three recurrent FT21 families, finding all three fathers (3/6 individuals; 50%) to be trisomy 21 mosaics; Pangalos et al. [30] found parental mosaicism in 5/13 phenotypically normal couples with recurrent FT21, that is 5/26 (19%) parents (one father and four mothers). In 1998, James et al. [39] found trisomy 21 mosaicism in 2/4 recurrent FT21 families, the affected parent being the mother (aged <35 years) in both cases. Parental mosaicism has been found to be the main cause of sibling recurrence FT21, and our data agree with this observation. Recurrent FT21 may occur in families without cytogenetically diagnosed parental mosaicism when the
33
trisomic cell line is confined to the germ cells or because of other etiological factors. The following causal factors other than parental mosaicism have been proposed [40–42]: (a) parental consanguinity-related genetic predisposition favoring non-disjunction; (b) chromosomal structural rearrangements distorting the meiotic process; (c) environmental predisposing factors; and (d) chance alone. No cases of parental consanguinity or chromosomal structural rearrangements were identified in our study. We observed parental trisomy 21 mosaicism in 2/44 (4.5%) individuals with only one FT21 product (Group II). Harris et al. [42] reviewed five cytogenetic surveys including a total of 200 couples with a single FT21 product; only 20–50 spreads were analyzed in most parents. Overall, 1.6% parents were identified as trisomy 21 mosaics, which is lower than the percentage reported here (4.5%). However, the analysis of 50 cells can only identify mosaics with ≥6–9% abnormal cells [32]. Our results and those previously reported in the medical literature suggest that parental mosaicism could be present in at least 3% of young couples with FT21 offspring, and when intentionally sought for, may be found as an important cause of FT21 (recurrent or isolated) in couples <35 years old [42,43]. Moreover, parental mosaicism may be part of the 5% of the paternal origin FT21 cases [6]. It is important to point out that in clinical practice, finding two cases of parental mosaicism among 44 parents of one FT21 product does not justify practicing the classical and molecular cytogenetic analyses described here in order to identify couples at high risk of conceiving a second DS product. This finding does, however, stress the need for prenatal diagnosis in all future pregnancies once a FT21 case has occurred. 4.3. Comparison of aneuploidy frequencies obtained by GTG-banding and FISH analyses The trisomy 21 frequencies obtained by GTGbanding were very similar to those obtained by FISH: 0.05 and 0.08 for Group I; 0.21 and 0.21 for Group II; 0.56 and 0.76 for Group III. Trisomy 13 cells were found only in individuals from Group II, with frequencies estimated at 0.007 by GTG-banding and 0.003 by FISH. Although trisomy 21 frequencies observed by both methods were very similar, the frequency of trisomy 13 according to FISH analysis was a half
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S. Frias et al. / Mutation Research 520 (2002) 25–37
of that observed on GTG-banded metaphase, but still within the range of frequencies of hyperdiploidy reported for autosomes [44]. We found that the total genome hyperdiploidy percentage was significantly higher (P < 0.05) when assessed by FISH than when assessed by GTG-banding (Tables 5 and 7). It was assumed that the frequency of non-disjunction is similar for all chromosomes to estimate this percentage, and this criterion is applied in most cases extrapolating data from one or a few chromosomes to the entire genome. However, according to previously reported data [45–49] and to our results, non-disjunction frequencies are not similar for all chromosomes (Table 5). Indeed, the highest trisomy frequencies involved chromosomes 21 and X, while we failed to identify trisomies involving chromosomes 1–8, 11, 12, 16, 18, 19 and Y among a total of 13,809 GTG banded cells. Thus, estimating total genome hyperdiploidy percentages based on data from only one or a few chromosomes is not useful to estimate the actual total genome trisomy frequency.
hyperdiploid cells (0.004–0.18% per chromosome) using conventional cytogenetic techniques. Considering 21, non-21 and marker chromosome trisomies, numerical chromosome abnormality frequencies were higher among the parents of one or more FT21 offspring (Groups II and III) as compared to the reference Group I (P < 0.05). Marker chromosomes were always supernumerary, implying the occurrence of at least two events: one altering the chromosome structure, and the other causing non-disjunction. This finding is in accordance with the report of Juberg et al. [10] who studied the frequency of hyperdiploidy in 21,841 cells of 717 individuals attending a cytogenetic diagnosis laboratory over a 5.4-year period. The authors found 15.8 hyperdiploid cells/2000 cells analyzed (0.79%) among parents of aneuploid offspring (the chromosomes involved were not specified), but only 2.56 hyperdiploid cells/2000 cells analyzed (0.13%) among the remainder individuals studied. The results of this and Juberg’s studies suggest that the frequency of aneuploidy is higher in parents of aneuploid offspring.
4.4. Frequency of hyperdiploid cells
4.5. Possible non-disjunction predisposing factors
Classical cytogenetic techniques allowed the observation of hyperdiploid cells involving trisomies for several non-21 chromosomes (Tables 5 and 6). The second most frequent trisomy involved the X chromosome (Table 5). It has been suggested that the X chromosome is more susceptible to mal-segregation than autosomes [50,51]. X chromosome aneuploidy has been frequently reported in mothers of DS children affecting 1.2–2% of the analyzed cells, and has been considered as a risk factor for DS offspring [10,29]. Several studies have proposed that non-disjunction involves chromosomes 21 and X more frequently than other chromosomes [10,47,50,51]. Our data are in agreement with these observations. This may occur because of positive selection of cells bearing these trisomies or higher mal-segregation frequencies for chromosomes X and 21. The hyperdiploidy frequencies found in couples with normal descendants (Group I; Tables 5 and 6) are similar to the frequencies previously reported by Eastmond and Pinkel [44] who found 0.5/1000 hyperdiploid cells using FISH probes for chromosomes 9 and X and Cimino et al. [45] who found 0.2/1000
Non-disjunction predisposing factors can be intrinsic or extrinsic [7,11,29,41,52,53]. Intrinsic factors such as constitutional heterochromatic polymorphisms [39], consanguinity, abnormal folate metabolism, genetic predisposition to non-disjunction and parental mosaicism, have been previously proposed [7,8,41,52]. In this study, aneuploidy frequencies were similar in individuals with and without polymorphisms, ruling out an interchromosomal effect. Moreover, all couples included in the study denied consanguinity, making the presence of an autosomal recessive gene responsible for predisposition to non-disjunction unlikely. Recombination frequency and advanced maternal age have been conclusively associated to FT21 [11,30]. We did in fact find evidence of an age effect on the frequency of aneuploidy, as previously reported [46–48]. Although all individuals were ≤35 years old and the mean age was very similar in all groups, the frequency of trisomy 21 and of other hyperdiploid cells was significantly higher in Group IIb (parents aged 25–35 years) than in Group IIa (parents aged 15–24 years; P < 0.05). However, on comparing
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Groups I and IIb, with no age differences (Table 6), hyperdiploid cell frequencies remained significantly higher in Group IIb. This suggests that although the effect of age is important, it is not the only factor involved. Extrinsic factor causing non-disjunction may be related to personal habits or environmental exposure. It is noteworthy that 20/26 Group II parents with hyperdiploidy were couples (both parents had aneuploidies in 10/13 couples), and 12/18 parents without hyperdiploidy were couples. Because none of these couples were consanguineous, this high concordance (16/22 concordant couples = 73%, binomial test P < 0.05) may be indicative of the role of extrinsic factors in the generation of aneuplidy. Personal habits including smoking, alcohol consumption, drug consumption or exposure to environmental factors such as radiation, have been proposed as possible causal factors of aneuploidy [52]. Because this study was not designed to determine causal factors, data are insufficient to draw any conclusions. However, it seems that factors such as the father’s profession or living in Mexico City metropolitan zone are unrelated to these events as all couples from Group I, and 20/25 couples with FT21 children lived in Mexico City. Only two couples from Group I admitted being smokers, and only one hyperdiploid cell was found in these smoking parents, while no hyperdiploid cells were observed in the only Group II couple who admitted smoking. Cigarette smoking has been considered as a potential etiological factor for DS [52]. Yang et al. [54] found a significant association between maternal smoking and FT21 with the effect confined to young mothers and MII cases. Other studies have failed to find this association or have even reported a negative association between smoking mothers and DS offspring. This be related with higher susceptibility of the DS product to the components of cigarette smoke, increasing the frequency of abortion of DS fetuses and, thus, diminishing the frequency of live DS births to smoking mothers [55–57]. However, it is known that cigarette smoke contains aneugenic agents and increases non-disjunction frequencies of specific chromosomes such as 13 and Y in sperm [57,58]. Specific studies should be designed to further investigate this candidate factor.
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Two of the DS mothers were asthmatic. This factor deserves further study, as certain bronchodilator medications such as theophylline are known to cause chromosomal aberrations. One of these asthmatic mothers (P4) had a high frequency of hyperdiploid metaphases and structural chromosomal aberrations (Tables 5 and 8); her clinical background included two fetal losses and one FT21 child. These data may be related with constant exposure to genotoxic environmental compounds and/or medications. Even though the sample size is very small, in Group II predisposition to non-disjunction or extrinsic factors may lead to a higher risk of hyperdiploid cells and DS offspring. Furthermore, these couples may also have a higher risk of unborn products affected with other non-21 trisomies. The high frequency of miscarriages among our FT21 couples (17 and 25% for Groups II and III, respectively) supports the latter statement. In clinical practice, the identification of a 21 trisomy cell line is of uttermost importance for appropriate genetic counseling; parental 21 trisomy mosaicism identified in peripheral lymphocytes makes prenatal diagnosis mandatory for future pregnancies. The precise recurrence risk for a mosaic individual depends on the percentage of trisomic cells in the gonads. Karyotype analyses, however, are mostly performed on somatic cells because of the difficulty in analyzing gonadal tissue [29,59,60]. Currently, aneuploidy can be identified in sperm by FISH analysis, and the technique will be available for clinic diagnosis in a near future [61,62]. The present study sought to analyze possible causal factors for FT21 cases born to young couples, as the majority of DS children are born to young parents. Up to date the relevance of parental mosaicism in the origin of FT21 has not been clearly established. Recognized etiological factors such as maternal age and recombination abnormalities do not explain the origin of most FT21 cases, so that several other factors must be causally involved. The role of extrinsic factors such as personal habits and environmental exposure remain to be determined. Acknowledgements The authors wish to thank Psic. Gerardo Barragán for his help with the statistical analysis and Dr. Ma. Teresa Villarreal for reviewing the language.
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References [1] F. Salamanca (Ed.), Citogenética Humana, 1a Edición, Editorial Médica Panamericana, Mexico, 1990, pp. 117–128. [2] B. Dagna, M. Pierluigi, M. Grasso, P. Strigini, Origin of extra chromosome 21 in 343 families: cytogenetic and molecular approaches, Am. J. Med. Suppl. 7 (1990) 129–132. [3] M. Nadal, S. Moreno, M. Pritchard, M.A. Preciado, DS: characterization of a case with partial trisomy of chromosome 21 owing to a paternal balanced translocation (15;21)(q26;q22.1) by FISH, J. Med. Genet. 34 (1997) 50–54. [4] F. Ballesta, R. Queralt, D. Gómez, E. Solsona, Parental origin and meiotic stage of non-disjunction in 139 cases of trisomy 21, Ann. Genet. 42 (1999) 11–15. [5] R. McKinlay, G. Sutherland (Eds.), Chromosome abnormalities and Genetic counseling, DS: Other Full Aneuploidies, and Polyploidy, Oxford University Press, New York, 1996, pp. 244–335. [6] M.B. Petersen, S.E. Antonarakis, T.J. Hassold, S.B. Freeman, S.L. Sherman, D. Avramopoulos, M. Mikkelsen, Paternal nondisjunction in trisomy 21: excess of male patients, Hum. Mol. Genet. 2 (1993) 1691–1695. [7] A. Savage, M. Petersen, D. Pettay, I. Taft, Elucidating the mechanisms of paternal non-disjinction of chromosome 21 in humans, Hum. Mol. Genet. 7 (1998) 1221–1227. [8] T. Hassold, M. Abruzzo, A. Kenneth, D. Griffin, Human aneuploidy: incidence, origin and etiology, Environ. Mol. Mutagen. 28 (1996) 167–175. [9] C.A. Huether, J. Ivanovich, B.S. Goodwin, E.L. Krivchenia, Maternal age specific risk rate estimates for DS among live births in whites and other races from Ohio and Metropolitan Atlanta, 1970–1989, J. Med. Genet. 35 (1998) 482–490. [10] R. Juberg, J. Knops, P. Mowrey, Increased frequency of lymphocytic mitotic non-disjunction in recurrent spontaneous aborters, J. Med. Genet. 22 (1985) 32–35. [11] M.B. Petersen, M. Mikkelsen, Nondisjunction in trisomy 21: origin and mechanisms, Cytogenet. Cell Genet. 91 (2000) 199–203. [12] S. Armendares, L. Buentello, F. Salamanca, Frecuencia de mixoploid´ıas en 85 casos ´ındice con s´ındrome de Down, Rev. Inv. Clin. 42 (1990) 103–107. [13] C.E. Blank, E. Gemmel, M.D. Casey, M. Lord, Mosaicism in a mother with a mongol child, Br. Med. J. 2 (1962) 378–380. [14] D.W. Smith, E.M. Therman, K.A. Patau, S.L. Inhorn, Mosaicism in mother with two mongols, Am. J. Dis. Child. 104 (1962) 534. [15] E.D. Weinstein, J. Warkany, Maternal mosaicism and Down’s syndrome (mongolism), J. Pediatr. 63 (1963) 599–604. [16] S. Ferier, Enfant mongolien-parent mosaique: étude de deux families, J. Genet. Hum. 13 (1964) 315–336. [17] H. Verresen, H. Van den Berghe, J. Creemers, Mosaic trisomy in the phenotypically normal mother of a mongol, Lancet 1 (1964) 526–527. [18] D. Aarskog, Down’s syndrome transmitted trough maternal mosaicism, Acta Paediatr. Scand. 58 (1969) 609–614. [19] M. Mikkelsen, A Danish survey of patients with Down’s syndrome born to young mothers, Ann. N. Y. Acad. Sci. 171 (1970) 370–378.
[20] E. Krmpotic, M.B. Hardin, Secondary nondisjunction causing regular trisomy 21 in the offspring of a mosaic trisomy 21 mother, Am. J. Obstet. Gynecol. 110 (1971) 589–590. [21] J. Timson, R. Harris, R.L. Gadd, M.E. Ferguson-Smith, Down’s syndrome due to maternal mosaicism and the value of antenatal diagnosis, Lancet 1 (1971) 549–550. [22] L. Hsu, M. Gertner, E. Leiter, K. Hirschhorn, Paternal trisomy 21 mosaicism and Down syndrome, Am. J. Hum. Genet. 23 (1971) 592–601. [23] K. Mehes, Paternal trisomy 21 mosaicism and Down’s anomaly, Humangenetik 17 (1973) 297–300. [24] S. Kaffe, L.Y.E. Hsu, K. Hirschhorn, Trisomy 21 mosaicism in a young woman with two children with trisomy 21 Down’ syndrome, J. Med. Genet. 11 (1974) 378–379. [25] Z. Papp, K. Cséscei, J. Skapinyerz, B. Dolhay, Paternal normal-trisomy 21 mosaicism as an indication for amniocentesis, Clin. Genet. 6 (1974) 192–194. [26] B.W. Richards, Investigation of 142 mosaic mongols and mosaic parents of mongols: cytogenetic analysis and maternal age at birth, J. Med. Def. Res. 18 (1974) 199–208. [27] F. Nuzzo, M. Stefanini, G. Simoni, A family with a three sibs carrying trisomy 21 mosaicism, Ann. Genet. 18 (1975) 111–116. [28] A. Osuna, A. Moreno, Regular G21-trisomy in 3 sibs from mother with trisomy 21 mosaicism, J. Med. Genet. 14 (1977) 286–287. [29] D.S. Krishna-Murthy, T.I. Farag, Recurrent regular trisomy 21 in two Bedouin families. Parental mosaicism versus genetic predisposition, Ann. Genet. 38 (1995) 217–224. [30] C. Pangalos, C.C. Talbot Jr., J.G. Lewis, P.A. Adeisberger, DNA polymorphism analysis in families with recurrence of free trisomy 21, Am. J. Hum. Genet. 51 (1992) 1027–1051. [31] J. Moorehead, P. Nowel, W. Mellman, D. Battips, Chromosome preparations of leucocytes cultured from human principal blood, Exp. Cell. Res. 20 (1960) 613–616. [32] E.B. Hook, Exclusion of chromosomal mosaicism: tables of 90, 95 and 99% confidence limits and comments on use, Am. J. Hum. Genet. 29 (1977) 94–97. [33] R. Lisker, Hallazgos citogenéticos en padecimientos hematológicos malignos, Rev. Invest. Clin. (Mex.) 39 (1987) 187–196. [34] S.L. Gersen, M.B. Keagle, Principles of clinical cytogenetics. Humana Press, NJ, 1999, pp. 73–76. [35] J. Anastasi, M. Le Beau, J. Vardiman, C. Westbrook, Detection of numerical chromosomal abnormalities in neoplastic hematopoietic cells by in situ hybridization with a chromosome-specific probe, Am. J. Pathol. 136 (1990) 131– 139. [36] A. Elhajouji, F. Tibaldi, M. Kirsch-Volders, Indication for a thresholds of chromosome non-disjuntion versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes, Mutagenesis 12 (1997) 133–140. [37] O.M. Mutchinik, R. Lisker, V. Babinski, Programa mexicano de registro y vigilancia epidemiológica de malformaciones congénitas externas, Salud Publica Méx. 30 (1988) 88–100. [38] D.K. Griffin, M.A. Abruzzo, E.A. Millie, E. Feingold, T.J. Hassold, Sex ratio in normal and disomic sperm: evidence that
S. Frias et al. / Mutation Research 520 (2002) 25–37
[39]
[40]
[41]
[42]
[43]
[44]
[45] [46]
[47]
[48]
[49]
[50]
the extra chromosome 21 preferentially segregates with the Y chromosome, Am. J. Hum. Genet. 59 (1996) 1108–1113. R.S. James, K. Ellis, D. Pettay, P.A. Jacobs, Cytogenetic and molecular study of four couples with multiple trisomy 21 pregnancies, Eur. J. Hum. Genet. 6 (1998) 207–212. S.K. Murthy, K. Prabhakara, Mitotic disturbance associated with inversion 9qh: a case report, Ann. Genet. 33 (1990) 169–172. O. Alfi, R. Chang, S. Azen, Evidence for genetic control of nondisjunction in man, Am. J. Hum. Genet. 32 (1980) 477– 483. D. Harris, M. Begleiter, J. Chamberlin, L. Hankis, Parental trisomy 21 mosaicism, Am. J. Hum. Genet. 34 (1982) 125– 133. E.S. Sachs, M.G.J. Jahoda, F.J. Los, L. Pijpers, J.W. Wladimiroff, Trisomy 21 mosaicism in gonads with unexpectedly high recurrence risks, Am. J. Med. Genet. 7 (1990) 186–188. D.A. Eastmond, D. Pinkel, Detection of aneuploidy and aneuploidy-inducing agents in human lymphocytes using fluorescence in situ hybridization with chromosome-specific DNA probes, Mutat. Res. 234 (1990) 303–318. M.C. Cimino, R.R. Tice, J.C. Liang, Aneuploidy in mammalian cells in vivo, Mutat. Res. 167 (1986) 107–122. P.A. Jacobs, W.M.C. Brown, Distribution of human chromosome counts in relation to age, Nature 191 (1961) 1178–1180. S.M. Galloway, K.E. Buckton, Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding, Cytogenet. Cell Genet. 20 (1978) 78–95. M.A. Bender, R.J. Preston, R.C. Leonard, B.E. Pyatt, P.C. Gooch, M.D. Shelby, Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample, Mutat. Res. 204 (1988) 421–433. L. Vega, M.E. Gonsebatt, P. Ostrosky-Wegman, Aneugenic effect of sodium arsenite on human lymphocytes in vitro: and individual susceptibility effect detected, Mutat. Res. 334 (1995) 365–373. A. Carere, A. Antoccia, D. Cimini, R. Crebelli, F. Degrassi, P. Leopardi, F. Marcon, A. Sgura, C. Tanzarella, A. Zijno, Análisis of chromosome loss and non-disjunction in cytokinesis-blocked lymphocytes of 24 male subjects, Mutagenesis 14 (1999) 491–496.
37
[51] J. Catalán, J. Surrallés, G.C.M. Falck, K. Autio, H. Norppa, Segregation of sex chromosomes in human lymphocytes, Mutagenesis 15 (2000) 251–255. [52] T. Hassold, P. Hunt, To err (meiotically) is human: the genesis of human aneuploidy, Nat. Rev. Genet. 2 (2001) 280– 291. [53] S. Antonarakis, S. Kittur, C. Metaxotou, P. Watkins, Analysis of DNA haplotypes suggests a genetic predisposition to trisomy 21 associated with DNA sequences on chrosome 21, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 3360–3364. [54] Q. Yang, S.L. Sherman, T.J. Hassold, K. Allran, L. Taft, D. Pettay, M.J. Khoury, J.D. Erickson, S.B. Freeman, Risk factors for trisomy 21: maternal cigarette smoking and oral contraceptive use in a population-based case–control study, Genet. Med. 1 (1999) 80–88. [55] C. Chen, T.J. Gilbert, J.R. Daling, Maternal smoking and Down syndrome: the confounding effect of maternal age, Am. J. Epidemiol. 149 (1999) 442–446. [56] E.B. Hook, P.K. Cross, Maternal cigarette smoking, Down syndrome in live births, and infant race, Am. J. Hum. Genet. 42 (1988) 482–489. [57] Q. Shi, E. Ko, L. Barclay, T. Hoang, A. Rademaker, R. Martin, Cigarette smoking and aneuploidy in human sperm, Mol. Reprod. Dev. 59 (2001) 417–421. [58] J. Rubes, X. Lowe, D. Moore II, S. Perreault, V. Slott, D. Evenson, S.G. Selevan, A.J. Wyrobek, Smoking cigarettes is associated with increased sperm disomy in teenage men, Fertil. Steril. 70 (1998) 715–723. [59] F. Vogel, A.G. Motulsky, Human genetics: problems and approaches, Mutation, Spnnger, Berlin, 1974, pp. 291–292, Chapter 5. [60] I. Uchida, V. Freeman, Trisomy 21 Down syndrome. II. Structural chromosome rearrangements in the parents, Hum. Genet. 72 (1986) 118–122. [61] A. Wyrobek, X.R. Lowe, Multiprobe fluorescence in situ hybridization methods for detecting chromosomally defective sperm in mice and humans, in: M. Andreeff, D. Pinkel (Eds.), Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications, Wiley, New York, 1999, pp. 391–408. [62] S. Frias, P. Van Hummelen, M. Meistrich, X. Lowe, M.D. Shelby, A.J. Wyrobek, NOVP chemotherapy induces transient increases in disomy 18 and 21 in sperm of Hodgkin’s disease patients, Am. J. Hum. Genet. S 63 (1998) 758.