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Down syndrome: a study of chromosomal mosaicism
RBMOnline - Vol 6. No 4. 499–503 Reproductive BioMedicine Online; www.rbmonline.com/Article/787 on web 17 February 2003
Article Down syndrome: a study of chromosomal mosaicism Deepak Modi obtained his Bachelors degree in Zoology in 1993 and obtained his Masters degree in Animal Physiology in 1995. He completed his PhD degree in Applied Biology in 2002, the subject of which was the understanding of the molecular basis of ovarian and testicular differentiation in human fetuses. His present research interests are in deciphering the molecular cascade of endometrial implantation and endometrial–embryo cross talk in primates, and the characterization of progesterone receptors on spermatozoa.
Dr Deepak Modi Deepak Modi1, Prajakta Berde, Deepa Bhartiya Cell Biology Department, Research Society, Bai Jerbai Wadia Hospital for Children, Acharya Donde Marg, Parel, Mumbai, India 1Correspondence: Primate Biology Division, National Institute for Research in Reproductive Health (NIRRH), JM Street, Parel, Mumbai 400 012, India. Tel: +91 22 4121111; e-mail: [email protected]
Abstract Recent data suggest that chromosome mosaicism is a possible mechanism for intrauterine and postnatal survival in cases of trisomy 18 and Turner syndrome (45X). The aim of this study was to evaluate if chromosomal mosaicism is a possible mechanism of survival in Down syndrome (DS) (trisomy 21) individuals. Mosaicism was studied by interphase fluorescence in-situ hybridization (FISH), using a specific probe for chromosome 21 (21q22.13–21q22.2) in 78 cases suspected of DS. To rule out tissue specific mosaicism, buccal cells or amniocytes were analysed in addition to blood in 20 DS cases. Thirtythree per cent of the cases studied by FISH in only peripheral blood were mosaics. In 20 cases of trisomy 21, two tissues were studied and mosaicism was not detected in either of the two tissues in 15 cases. The remaining five cases were mosaics in both the tissues analysed. Clinical comparisons in 17 DS mosaics showed a direct relationship between the percentage of trisomic cells and the degree of phenotypic manifestations. These results suggest that mechanism(s) other than mosaicism may exist for the intrauterine and postnatal survival of DS cases. Keywords: chromosome aneuploidy, Down syndrome, fluorescence in-situ hybridization (FISH), mosaicism, trisomy 21
Introduction At least 25% of preimplantation conceptuses and 15% of clinically recognized pregnancies are lost spontaneously. One major reason for this high rate of pregnancy loss is chromosome aneuploidy. A significant proportion of human preimplantation embryos and first trimester fetuses that abort spontaneously are chromosomally defective (Jacobs and Hassold, 1987; Márquez et al., 2000; Munné et al., 2002). Approximately 50% of first trimester spontaneous abortions are trisomic, 20% are monosomic and 25% are polyploid (Jacobs and Hassold, 1987). Trisomy for almost every chromosome has been described in spontaneously aborted fetuses; some trisomies are very frequent (e.g. trisomy 15, 16, 21, 18), whereas others are rare (e.g. trisomy 1, 5, 19). Irrespective of their frequency of occurrence, one common feature of all trisomies is that they act as lethal mutations to the developing embryo or fetus, although some trisomic fetuses do survive to term. It has been
estimated that of conceptuses with trisomy 13 or 18, less than 5% survive till term, whereas about 20–25% of trisomy 21 conceptuses are live born (Kalousek et al., 1989). What makes these few trisomic conceptions different from others in terms of their intrauterine survival? Current dogma holds that under the pressure of natural selection, most aneuploid conceptions are aborted spontaneously, but natural selection does not prevail when mosaicism is operative; thus most live born chromosomally aneuploid fetuses may be mosaics (Hook and Warburton, 1983; Kalousek et al., 1989; Modi et al., 1999). Using interphase fluorescence in-situ hybridization (FISH), it has been shown that all live born cases of trisomy 18 have a coexisting normal diploid (disomy 18) cell line, indicating that mosaicism is a possible mechanism of intrauterine and postnatal survival of infants with trisomy 18 (Modi et al., 1999). However, in the same study, only 20% of Down syndrome (DS) patients studied were mosaics. This frequency of mosaicism was lower than that observed in cases of trisomy 18 (100%) and Turner syndrome (75%) (Modi et
499
Articles - Mosaicism in Down syndrome - D Modi et al.
al., 1999). It was concluded that chromosome mosaicism might not be a possible mechanism for survival of DS infants. However, in that study, only peripheral blood cells were analysed for detection of mosaicism. It is likely that the absence of mosaicism in blood does not imply its absence in other tissues, as FISH analysis in multiple tissues of Turner syndrome fetuses revealed mosaicism in some tissues that was not detected in amniotic fluid cells (Ameil et al., 1996). Similarly, it is possible that live born cases of DS may be mosaics in tissues other than blood. Another possible reason for the low incidence of trisomy 21 mosaics observed in the earlier study (Modi et al., 1999) could be the failure to detect mosaicism because of technical difficulties. In the previous study, an alpha satellite probe was used to detect trisomy 21; through sequence homology, the probe also cross hybridizes to the centromere of chromosome 13, yielding four hybridization signals in a normal case. This probe has limited sensitivity in detecting low level mosaicism as compared with other chromosome specific probes (Modi et al., 1999). Hence it is likely that mosaicism, particularly of low degree, may have been missed. Therefore the purpose of the study was to analyse the ploidy level of chromosome 21 in clinically suspected cases of DS using a probe specific to chromosome 21. To rule out tissue specific mosaicism cells from buccal mucosa or amniocytes were analysed in addition to peripheral blood. The aim of the study was to detect trisomy 21 mosaicism more sensitively and hence comment on whether or not mosaicism is a possible mechanism of intrauterine survival of trisomy 21 infants.
Materials and methods Seventy-eight cases with a clinical suspicion of DS were included in this study. The inclusion criteria were those described previously (Modi et al., 1999). Mononuclear cells from peripheral blood or cord blood were isolated and fixed according to standard cytogenetic protocol. Oral mucosa cells (buccal cells) were washed twice in normal saline and fixed in 3:1 methanol:acetic acid. Aliquots of 15 ml of amniotic fluid were collected transabdominally (16–24 weeks of gestation) and spun at 500 g for 10 min. The pellet (amniocytes) was
500
washed once in normal saline and processed according to routine cytogenetic protocol. The specific probe for chromosome 21 was purchased from Vysis (Richmond, UK), and encompassed D21S259. D21S341 and D21S342 loci on the long arm of chromosome 21 (21q22.13–21q22.2). FISH was performed according to manufacturer’s instructions. Briefly, the cells were spread on a silane-treated slide and airdried. Amniocytes and buccal cells were pretreated with 0.005% pepsin and post-fixed in 4% formaldehyde. After incubation in 2× SSC at 37°C, the slides were dehydrated in ethanol grades. The diluted probe was then applied on the slides, sealed and co-denatured with the target cells at 75°C for 5 min. Hybridization was performed overnight at 37°C. The slides were washed in 0.4× SSC at 70°C for 2 min and in 2× SSC at room temperature for 1 min. The cells were mounted in aquamount containing an antifade and DAPI (4′,6-diamido-2phenylindole) as a counterstain. At least 500 mononuclear cells, 200 buccal cells or 100 amniocytes were scored for the number of signals under a fluorescence microscope (Olympus BX 60) using appropriate filter sets. Mononuclear cells, buccal cells and amniotic fluid cells from 25 normal cases were analysed to detect hybridization efficiency and frequency of signal distribution.
Results The hybridization efficiency of the probe used was 99% for mononuclear and buccal cells and 96% for amniocytes. The lower limit for detecting a separate cell line was 2% in mononuclear and buccal cells and 4% for amniocytes. These values corresponded to the mean +2 SD of the false positive as reported previously (Ameil et al., 1996; Modi et al., 1999). As the true false negative values cannot be estimated, the upper limit for detecting mosaicism was arbitrarily assumed as 95%. This implied that any case showing <2% trisomic cells in blood/buccal cells and <4% in amniocytes was considered as normal and a non-mosaic trisomy was defined when >95% cells were trisomic. Figure 1 shows FISH results of a case with trisomy 21 mosaicism detected in the peripheral blood. Seventy of 78 cases with clinical suspicion of DS were found to be trisomic
Figure 1: Fluorescence in-situ hybridization (FISH) on blood mononuclear cells using a locus specific 21 chromosome probe. Note the presence of two and three signals (arrow) indicating mosaicism.
Articles - Mosaicism in Down syndrome - D Modi et al.
Table 1. Details of live born Down syndrome cases selected in the present study. No. of cases selected No. of cases affected No. of mosaics (%) Age range Sex ratio (male:female)
Table 2. Comparison of the percentage of trisomic cells detected by FISH in mononuclear cells from the blood and buccal cells or amniocytes in trisomy 21 patients.
78 70 23 (33) 2 days to 6 years 4:1
Serial no.
Blood
1 2 3 4 5 6
in peripheral blood, of which 33% (n = 23) were mosaics (Table 1). Blood and buccal cells or amniocytes were examined in only 20 cases of DS (Table 2). Of these, 5/20 cases were mosaics in both the tissues studied. Mosaicism was not detected in any of the two tissues in the remaining 15 cases. The extent of mosaicism (percent trisomic cells) varied between the two tissues in cases 1, 2 and 3. However, in the remaining cases (two mosaic and 15 non-mosaics), the numbers of trisomic cells were not significantly different in the two cell types studied.
50 15 18 35 50 99
36 28 10 25 45 100
100 100
98 100
10 11 12
100 99 100 100
96 100 100 98
13a 14 15 16 17 18 19 20
99 97 100 100 97 99 99 100
95 98 95 100 98 95 96 97
7a 8 9a
Table 3 gives the phenotypic characteristics of 17 mosaic DS cases. There is an apparent correspondence between the number of trisomic cells and the phenotypic manifestations (Table 4); the number of phenotypes appears to reduce with the fall in the number of trisomic cells (r2 = 0.602; statistical test not applied because of low numbers). The small size of the sample, together with the subjective nature of scoring DS phenotypes quantitatively, precluded a more detailed statistical analysis.
Buccal cells or amniocytes
aAmniocytes.
Table 3. Phenotypic characteristics of 17 mosaic DS cases. Percentage trisomy
90
80
70
60
50
25
18
10
Patient no.
1
2
1
2
1
2
1
2
1
2
3
1
2
3
1
1
2
Hypotonia Clinodactly Wide gap between 1st and 2nd toes Broad short hands Simian crease Protruding tongue Fissured tongue Low set/small ears Depressed nasal bridge Small nose Epicanthic folds Flat facies Upward slant of eyes Accessory 3rd fontanelle Brachycephaly Microcephaly Cardiac anomaly
+ + +
+ + +
+ – –
+ + +
+ + –
+ + +
+ + –
+ + –
+ – –
+ + –
+ + +
– – –
+ + +
– + –
– – –
– + +
– + +
+ + + – + +
+ + + + + +
+ + + – + +
+ + + – + +
+ – + + + –
? + + + + +
+ – – – – +
+ + + – + +
– + – – – +
+ ? + – + +
+ + + – + +
+ – – – + +
+ – + – – +
+ + – – + +
– – – – + –
– – – – – +
– – – – – +
+ + + +
– + + +
+ + + –
– + + –
+ + + +
+ + + +
+ – + +
– + – –
– – + +
+ + + +
+ + – –
– – + –
– ? + –
+ + + –
– – – +
– + – +
– – – –
?
+
–
+
–
+
–
–
–
?
+
–
–
–
–
–
–
– + –
– + +
? – –
+ – –
+ – –
+ – +
– + –
– + +
– – +
+ + ?
– – +
– – –
+ + +
– + –
– + –
– – –
– + +
+ = present; – = absent; ? = unknown/not recorded.
501
Articles - Mosaicism in Down syndrome - D Modi et al.
Table 4. Numerical relationship between percentage of trisomic cellsa and number of phenotypes in mosaic DS casesa. Trisomic cells (%)
Number of phenotypes present
90 90 80 80 70 70 60 60 50 50 50 25 25 25 18 10 10
13 15 9 12 11 15 8 10 6 12 11 4 10 9 3 5 5
aData derived from Table 3.
Discussion In all, 33% of DS patients included in the present study showed mosaicism using a FISH probe specific for chromosome 21. This number is higher than that reported earlier (20%) (Modi et al., 1999) using a common alpha satellite 13/21 probe (20%). It is interesting to note that the frequency of mosaicism as detected by FISH is comparatively higher in cases of Turner syndrome (75%) and trisomy 18 (100%) than that observed for DS in the present study (Fernandez et al., 1996; Modi et al., 1999). Thus, it appears that chromosomal mosaicism is possibly not a common phenomenon in cases of Down syndrome. However, absence of mosaicism in blood does not exclude mosaicism in other tissues. Tissue specific mosaicism has been reported in cases of trisomy 18, trisomy 13 and Turner syndrome (Kalousek et al., 1989; Ameil et al., 1996; D Modi, unpublished data). To verify this possibility, buccal cells or amniotic fluid were analysed by FISH in 20 cases of DS and the results were compared with those obtained from blood. As evident from Table 2, the degree of mosaicism varied between the two tissues in some mosaic individuals, but none of the non-mosaic cases (in blood) was found to be mosaic in the alternate tissue investigated. Mosaicism was not detected by FISH in the cells obtained from urine of four non-mosaic cases of DS (D Modi, unpublished data). These findings corroborate the results of Kalousek et al. (1989), who did not detect mosaicism in any of the embryonic or extra-embryonic tissues of trisomy 21 newborns. Thus, it appears that mosaicism is probably not a common event in cases of trisomy 21, and it is tempting to speculate that a mechanism other than mosaicism exists to facilitate the survival of these fetuses.
502
Chromosome 21 is one of the smallest chromosomes in the human genome. Recent sequencing data have shown that chromosome 21 contains approximately 225 genes in the 33.8
Mb region sequenced (Hattori et al., 2000). However, in comparison, in chromosome 22, which is the same size as chromosome 21, the 33.4 Mb region sequenced has 545 genes (Dunham et al., 1999). These results imply that chromosome 21 is gene poor as compared with chromosome 22. Thus, it is possible that due to the low density of genes on chromosome 21, trisomy 21 is one of the most common viable non-mosaic autosomal trisomies. The survival of an embryo largely depends on a favourable intrauterine anatomical, biochemical and physiological milieu. It has been speculated that the intrauterine milieu buffers the environmental insults and stochastic errors that the embryo faces during development, thereby preventing the development of serious birth defects (Shapiro, 1989, 1994). Furthermore, it has been suggested that the loss of genetic balance (e.g. a trisomy) predisposes the embryo to the traumatic factors that lead to the formation of congenital malformations and probably even embryo death (Shapiro, 1989, 1994). It has been previously shown that maternal factors such as race, fever, alcohol use and exposure to cigarette smoke during pregnancy influences the phenotypic manifestations in DS (Khoury and Erickson, 1992). Thus, developmental instability and altered homeostasis of the aneuploid embryos increase its susceptibility to the environmental factors, resulting in serious malformations and even death. In this context, it is possible that DS fetuses (although aneuploid) are less susceptible to the environmental insults and stochastic errors faced in utero as compared with other aneuploid individuals (e.g. trisomy 18, monosomy X), resulting in the intrauterine and postnatal survival of these infants even in absence of mosaicism. However, this hypothesis needs to be exhaustively tested. In accordance with previous results in cases of trisomy 18 (Modi et al., 1999), in the present study too, a relationship was found between the number of trisomic cells in blood and the phenotypic manifestations in DS cases. Although the numbers of cases studied are too limited for rigorous statistical analysis, a positive correlation (r2 = 0.602) between the number of trisomic cells and the phenotypic expression was observed. Maximum phenotypes were noted when the number of trisomic cells in blood was >50%, whereas the number of phenotypes was minimal in mosaic individuals where the percentage of trisomic cells was <20%. These results further confirm the long-existing notion that mosaic aneuploid individuals are clinically advantaged over non-mosaics (Benda, 1969; Percy et al., 1993). However, detailed clinical assessment and long-term follow-up of these cases will be required for better understanding of this relationship and making use of these findings for counselling the affected patients especially mosaics. The results here also recommend the use of sensitive molecular techniques such as interphase FISH in clinical practice for diagnosis of chromosomal disorders, as FISH permits identification of the aneuploidy even when it is present in low grade (low level mosaicism) which may not be detected by using conventional karyotyping. This has particular relevance in terms of clinical management of the patients and counselling of the parents, especially for prenatal diagnosis in the next pregnancy.
Articles - Mosaicism in Down syndrome - D Modi et al.
In conclusion, the frequency of mosaicism as studied by FISH in DS individuals is greater (33%) than that reported previously (20%). However, since this frequency is still lower than that reported for trisomy 18 (100%) and Turner syndrome cases (75%), it is likely that different fetoprotective pathway(s) exist which facilitate the survival of DS individuals.
Acknowledgements This work was supported by a grant from the Department of Biotechnology, Ministry of Health and Sciences, India. We thank Dr Sudha G Gangal (Director) for her valuable suggestions. We are very grateful to the clinical staff of our Genetic clinic.
References Ameil A, Kidron D, Kedar I et al. 1996 Are all phenotypically normal Turner fetuses mosaics? Prenatal Diagnosis 19, 791–795. Benda CE 1969 Down’s syndrome. Mongolism and its Management. Grune and Stratton, New York, pp. 121–133. Dunham I, Collins JE, Bruskiewich R et al. 1999 The DNA sequence of chromosome 22. Nature 402, 489–495. Fernandez R, Mendez J, Pasaro E 1996 Turner syndrome: a study of chromosomal mosaicism. Human Genetics 98, 29–35. Hattori M, Fujiyama A, Taylor TD et al. 2000 The DNA sequence of human chromosome 21. Nature 405, 311–319. Hook EB, Warburton D 1983 The distribution of chromosomal genotypes associated with Turner’s syndrome: live birth prevalence and evidence for dismissed fetal mortality and severity in genotypes associated with structural abnormalities or mosaicism. Human Genetics 64, 24–27.
Jacobs P, Hassold T 1987 Chromosome abnormalities: etiology in abortions and livebirths. In: Vogel F, Sperling K (eds) Human Genetics. Springer, Berlin and Heidelberg, pp. 233–245. Kalousek DK, Barrett IJ, McGillivray BC 1989 Placental mosaicism and intrauterine survival of trisomies 13 and 18. American Journal of Human Genetics 44, 338–343. Khoury MJ, Erickson LD 1992 Can maternal risk factors influence the presence of major birth defects in infants with Down syndrome? American Journal of Medical Genetics 43, 1016–1022. Márquez C, Sandalinas M, Bahce M et al. 2000 Chromosome abnormalities in 1255 cleavage-stage human embryos. Reproductive BioMedicine Online 1, 17–27. Modi D, Kher A, Parikh A, Bhartiya D 1999 Chromosomal mosaicism: evaluation by fluorescence in situ hybridization. Medical Science Research 27, 813–815. Munné S, Sandalinas M, Escudero T et al. 2002 Chromosome mosaicism in cleavage-stage human embryos: evidence for a maternal age effect. Reproductive BioMedicine Online 4, 223–232. Percy ME, Markovic VD, Dalton AJ et al. 1993 Age associated chromosome 21 loss in Down syndrome: possible relevance to mosaicism and Alzheimer disease. American Journal of Medical Genetics 45, 584–588. Shapiro BL 1989 The pathogenesis of aneuploid phenotypes: the fallacy of explanatory reductionism. American Journal of Medical Genetics 33, 146–150. Shapiro BL 1994 The environmental basis of the Down syndrome phenotype. Developmental Medicine and Child Neurology 36, 84–90. Received 14 October 2002; refereed 1 November 2002; accepted 13 January 2003.
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Article Down syndrome: a study of chromosomal mosaicism Deepak Modi obtained his Bachelors degree in Zoology in 1993 and obtained his Masters degree in Animal Physiology in 1995. He completed his PhD degree in Applied Biology in 2002, the subject of which was the understanding of the molecular basis of ovarian and testicular differentiation in human fetuses. His present research interests are in deciphering the molecular cascade of endometrial implantation and endometrial–embryo cross talk in primates, and the characterization of progesterone receptors on spermatozoa.
Dr Deepak Modi Deepak Modi1, Prajakta Berde, Deepa Bhartiya Cell Biology Department, Research Society, Bai Jerbai Wadia Hospital for Children, Acharya Donde Marg, Parel, Mumbai, India 1Correspondence: Primate Biology Division, National Institute for Research in Reproductive Health (NIRRH), JM Street, Parel, Mumbai 400 012, India. Tel: +91 22 4121111; e-mail: [email protected]
Abstract Recent data suggest that chromosome mosaicism is a possible mechanism for intrauterine and postnatal survival in cases of trisomy 18 and Turner syndrome (45X). The aim of this study was to evaluate if chromosomal mosaicism is a possible mechanism of survival in Down syndrome (DS) (trisomy 21) individuals. Mosaicism was studied by interphase fluorescence in-situ hybridization (FISH), using a specific probe for chromosome 21 (21q22.13–21q22.2) in 78 cases suspected of DS. To rule out tissue specific mosaicism, buccal cells or amniocytes were analysed in addition to blood in 20 DS cases. Thirtythree per cent of the cases studied by FISH in only peripheral blood were mosaics. In 20 cases of trisomy 21, two tissues were studied and mosaicism was not detected in either of the two tissues in 15 cases. The remaining five cases were mosaics in both the tissues analysed. Clinical comparisons in 17 DS mosaics showed a direct relationship between the percentage of trisomic cells and the degree of phenotypic manifestations. These results suggest that mechanism(s) other than mosaicism may exist for the intrauterine and postnatal survival of DS cases. Keywords: chromosome aneuploidy, Down syndrome, fluorescence in-situ hybridization (FISH), mosaicism, trisomy 21
Introduction At least 25% of preimplantation conceptuses and 15% of clinically recognized pregnancies are lost spontaneously. One major reason for this high rate of pregnancy loss is chromosome aneuploidy. A significant proportion of human preimplantation embryos and first trimester fetuses that abort spontaneously are chromosomally defective (Jacobs and Hassold, 1987; Márquez et al., 2000; Munné et al., 2002). Approximately 50% of first trimester spontaneous abortions are trisomic, 20% are monosomic and 25% are polyploid (Jacobs and Hassold, 1987). Trisomy for almost every chromosome has been described in spontaneously aborted fetuses; some trisomies are very frequent (e.g. trisomy 15, 16, 21, 18), whereas others are rare (e.g. trisomy 1, 5, 19). Irrespective of their frequency of occurrence, one common feature of all trisomies is that they act as lethal mutations to the developing embryo or fetus, although some trisomic fetuses do survive to term. It has been
estimated that of conceptuses with trisomy 13 or 18, less than 5% survive till term, whereas about 20–25% of trisomy 21 conceptuses are live born (Kalousek et al., 1989). What makes these few trisomic conceptions different from others in terms of their intrauterine survival? Current dogma holds that under the pressure of natural selection, most aneuploid conceptions are aborted spontaneously, but natural selection does not prevail when mosaicism is operative; thus most live born chromosomally aneuploid fetuses may be mosaics (Hook and Warburton, 1983; Kalousek et al., 1989; Modi et al., 1999). Using interphase fluorescence in-situ hybridization (FISH), it has been shown that all live born cases of trisomy 18 have a coexisting normal diploid (disomy 18) cell line, indicating that mosaicism is a possible mechanism of intrauterine and postnatal survival of infants with trisomy 18 (Modi et al., 1999). However, in the same study, only 20% of Down syndrome (DS) patients studied were mosaics. This frequency of mosaicism was lower than that observed in cases of trisomy 18 (100%) and Turner syndrome (75%) (Modi et
499
Articles - Mosaicism in Down syndrome - D Modi et al.
al., 1999). It was concluded that chromosome mosaicism might not be a possible mechanism for survival of DS infants. However, in that study, only peripheral blood cells were analysed for detection of mosaicism. It is likely that the absence of mosaicism in blood does not imply its absence in other tissues, as FISH analysis in multiple tissues of Turner syndrome fetuses revealed mosaicism in some tissues that was not detected in amniotic fluid cells (Ameil et al., 1996). Similarly, it is possible that live born cases of DS may be mosaics in tissues other than blood. Another possible reason for the low incidence of trisomy 21 mosaics observed in the earlier study (Modi et al., 1999) could be the failure to detect mosaicism because of technical difficulties. In the previous study, an alpha satellite probe was used to detect trisomy 21; through sequence homology, the probe also cross hybridizes to the centromere of chromosome 13, yielding four hybridization signals in a normal case. This probe has limited sensitivity in detecting low level mosaicism as compared with other chromosome specific probes (Modi et al., 1999). Hence it is likely that mosaicism, particularly of low degree, may have been missed. Therefore the purpose of the study was to analyse the ploidy level of chromosome 21 in clinically suspected cases of DS using a probe specific to chromosome 21. To rule out tissue specific mosaicism cells from buccal mucosa or amniocytes were analysed in addition to peripheral blood. The aim of the study was to detect trisomy 21 mosaicism more sensitively and hence comment on whether or not mosaicism is a possible mechanism of intrauterine survival of trisomy 21 infants.
Materials and methods Seventy-eight cases with a clinical suspicion of DS were included in this study. The inclusion criteria were those described previously (Modi et al., 1999). Mononuclear cells from peripheral blood or cord blood were isolated and fixed according to standard cytogenetic protocol. Oral mucosa cells (buccal cells) were washed twice in normal saline and fixed in 3:1 methanol:acetic acid. Aliquots of 15 ml of amniotic fluid were collected transabdominally (16–24 weeks of gestation) and spun at 500 g for 10 min. The pellet (amniocytes) was
500
washed once in normal saline and processed according to routine cytogenetic protocol. The specific probe for chromosome 21 was purchased from Vysis (Richmond, UK), and encompassed D21S259. D21S341 and D21S342 loci on the long arm of chromosome 21 (21q22.13–21q22.2). FISH was performed according to manufacturer’s instructions. Briefly, the cells were spread on a silane-treated slide and airdried. Amniocytes and buccal cells were pretreated with 0.005% pepsin and post-fixed in 4% formaldehyde. After incubation in 2× SSC at 37°C, the slides were dehydrated in ethanol grades. The diluted probe was then applied on the slides, sealed and co-denatured with the target cells at 75°C for 5 min. Hybridization was performed overnight at 37°C. The slides were washed in 0.4× SSC at 70°C for 2 min and in 2× SSC at room temperature for 1 min. The cells were mounted in aquamount containing an antifade and DAPI (4′,6-diamido-2phenylindole) as a counterstain. At least 500 mononuclear cells, 200 buccal cells or 100 amniocytes were scored for the number of signals under a fluorescence microscope (Olympus BX 60) using appropriate filter sets. Mononuclear cells, buccal cells and amniotic fluid cells from 25 normal cases were analysed to detect hybridization efficiency and frequency of signal distribution.
Results The hybridization efficiency of the probe used was 99% for mononuclear and buccal cells and 96% for amniocytes. The lower limit for detecting a separate cell line was 2% in mononuclear and buccal cells and 4% for amniocytes. These values corresponded to the mean +2 SD of the false positive as reported previously (Ameil et al., 1996; Modi et al., 1999). As the true false negative values cannot be estimated, the upper limit for detecting mosaicism was arbitrarily assumed as 95%. This implied that any case showing <2% trisomic cells in blood/buccal cells and <4% in amniocytes was considered as normal and a non-mosaic trisomy was defined when >95% cells were trisomic. Figure 1 shows FISH results of a case with trisomy 21 mosaicism detected in the peripheral blood. Seventy of 78 cases with clinical suspicion of DS were found to be trisomic
Figure 1: Fluorescence in-situ hybridization (FISH) on blood mononuclear cells using a locus specific 21 chromosome probe. Note the presence of two and three signals (arrow) indicating mosaicism.
Articles - Mosaicism in Down syndrome - D Modi et al.
Table 1. Details of live born Down syndrome cases selected in the present study. No. of cases selected No. of cases affected No. of mosaics (%) Age range Sex ratio (male:female)
Table 2. Comparison of the percentage of trisomic cells detected by FISH in mononuclear cells from the blood and buccal cells or amniocytes in trisomy 21 patients.
78 70 23 (33) 2 days to 6 years 4:1
Serial no.
Blood
1 2 3 4 5 6
in peripheral blood, of which 33% (n = 23) were mosaics (Table 1). Blood and buccal cells or amniocytes were examined in only 20 cases of DS (Table 2). Of these, 5/20 cases were mosaics in both the tissues studied. Mosaicism was not detected in any of the two tissues in the remaining 15 cases. The extent of mosaicism (percent trisomic cells) varied between the two tissues in cases 1, 2 and 3. However, in the remaining cases (two mosaic and 15 non-mosaics), the numbers of trisomic cells were not significantly different in the two cell types studied.
50 15 18 35 50 99
36 28 10 25 45 100
100 100
98 100
10 11 12
100 99 100 100
96 100 100 98
13a 14 15 16 17 18 19 20
99 97 100 100 97 99 99 100
95 98 95 100 98 95 96 97
7a 8 9a
Table 3 gives the phenotypic characteristics of 17 mosaic DS cases. There is an apparent correspondence between the number of trisomic cells and the phenotypic manifestations (Table 4); the number of phenotypes appears to reduce with the fall in the number of trisomic cells (r2 = 0.602; statistical test not applied because of low numbers). The small size of the sample, together with the subjective nature of scoring DS phenotypes quantitatively, precluded a more detailed statistical analysis.
Buccal cells or amniocytes
aAmniocytes.
Table 3. Phenotypic characteristics of 17 mosaic DS cases. Percentage trisomy
90
80
70
60
50
25
18
10
Patient no.
1
2
1
2
1
2
1
2
1
2
3
1
2
3
1
1
2
Hypotonia Clinodactly Wide gap between 1st and 2nd toes Broad short hands Simian crease Protruding tongue Fissured tongue Low set/small ears Depressed nasal bridge Small nose Epicanthic folds Flat facies Upward slant of eyes Accessory 3rd fontanelle Brachycephaly Microcephaly Cardiac anomaly
+ + +
+ + +
+ – –
+ + +
+ + –
+ + +
+ + –
+ + –
+ – –
+ + –
+ + +
– – –
+ + +
– + –
– – –
– + +
– + +
+ + + – + +
+ + + + + +
+ + + – + +
+ + + – + +
+ – + + + –
? + + + + +
+ – – – – +
+ + + – + +
– + – – – +
+ ? + – + +
+ + + – + +
+ – – – + +
+ – + – – +
+ + – – + +
– – – – + –
– – – – – +
– – – – – +
+ + + +
– + + +
+ + + –
– + + –
+ + + +
+ + + +
+ – + +
– + – –
– – + +
+ + + +
+ + – –
– – + –
– ? + –
+ + + –
– – – +
– + – +
– – – –
?
+
–
+
–
+
–
–
–
?
+
–
–
–
–
–
–
– + –
– + +
? – –
+ – –
+ – –
+ – +
– + –
– + +
– – +
+ + ?
– – +
– – –
+ + +
– + –
– + –
– – –
– + +
+ = present; – = absent; ? = unknown/not recorded.
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Articles - Mosaicism in Down syndrome - D Modi et al.
Table 4. Numerical relationship between percentage of trisomic cellsa and number of phenotypes in mosaic DS casesa. Trisomic cells (%)
Number of phenotypes present
90 90 80 80 70 70 60 60 50 50 50 25 25 25 18 10 10
13 15 9 12 11 15 8 10 6 12 11 4 10 9 3 5 5
aData derived from Table 3.
Discussion In all, 33% of DS patients included in the present study showed mosaicism using a FISH probe specific for chromosome 21. This number is higher than that reported earlier (20%) (Modi et al., 1999) using a common alpha satellite 13/21 probe (20%). It is interesting to note that the frequency of mosaicism as detected by FISH is comparatively higher in cases of Turner syndrome (75%) and trisomy 18 (100%) than that observed for DS in the present study (Fernandez et al., 1996; Modi et al., 1999). Thus, it appears that chromosomal mosaicism is possibly not a common phenomenon in cases of Down syndrome. However, absence of mosaicism in blood does not exclude mosaicism in other tissues. Tissue specific mosaicism has been reported in cases of trisomy 18, trisomy 13 and Turner syndrome (Kalousek et al., 1989; Ameil et al., 1996; D Modi, unpublished data). To verify this possibility, buccal cells or amniotic fluid were analysed by FISH in 20 cases of DS and the results were compared with those obtained from blood. As evident from Table 2, the degree of mosaicism varied between the two tissues in some mosaic individuals, but none of the non-mosaic cases (in blood) was found to be mosaic in the alternate tissue investigated. Mosaicism was not detected by FISH in the cells obtained from urine of four non-mosaic cases of DS (D Modi, unpublished data). These findings corroborate the results of Kalousek et al. (1989), who did not detect mosaicism in any of the embryonic or extra-embryonic tissues of trisomy 21 newborns. Thus, it appears that mosaicism is probably not a common event in cases of trisomy 21, and it is tempting to speculate that a mechanism other than mosaicism exists to facilitate the survival of these fetuses.
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Chromosome 21 is one of the smallest chromosomes in the human genome. Recent sequencing data have shown that chromosome 21 contains approximately 225 genes in the 33.8
Mb region sequenced (Hattori et al., 2000). However, in comparison, in chromosome 22, which is the same size as chromosome 21, the 33.4 Mb region sequenced has 545 genes (Dunham et al., 1999). These results imply that chromosome 21 is gene poor as compared with chromosome 22. Thus, it is possible that due to the low density of genes on chromosome 21, trisomy 21 is one of the most common viable non-mosaic autosomal trisomies. The survival of an embryo largely depends on a favourable intrauterine anatomical, biochemical and physiological milieu. It has been speculated that the intrauterine milieu buffers the environmental insults and stochastic errors that the embryo faces during development, thereby preventing the development of serious birth defects (Shapiro, 1989, 1994). Furthermore, it has been suggested that the loss of genetic balance (e.g. a trisomy) predisposes the embryo to the traumatic factors that lead to the formation of congenital malformations and probably even embryo death (Shapiro, 1989, 1994). It has been previously shown that maternal factors such as race, fever, alcohol use and exposure to cigarette smoke during pregnancy influences the phenotypic manifestations in DS (Khoury and Erickson, 1992). Thus, developmental instability and altered homeostasis of the aneuploid embryos increase its susceptibility to the environmental factors, resulting in serious malformations and even death. In this context, it is possible that DS fetuses (although aneuploid) are less susceptible to the environmental insults and stochastic errors faced in utero as compared with other aneuploid individuals (e.g. trisomy 18, monosomy X), resulting in the intrauterine and postnatal survival of these infants even in absence of mosaicism. However, this hypothesis needs to be exhaustively tested. In accordance with previous results in cases of trisomy 18 (Modi et al., 1999), in the present study too, a relationship was found between the number of trisomic cells in blood and the phenotypic manifestations in DS cases. Although the numbers of cases studied are too limited for rigorous statistical analysis, a positive correlation (r2 = 0.602) between the number of trisomic cells and the phenotypic expression was observed. Maximum phenotypes were noted when the number of trisomic cells in blood was >50%, whereas the number of phenotypes was minimal in mosaic individuals where the percentage of trisomic cells was <20%. These results further confirm the long-existing notion that mosaic aneuploid individuals are clinically advantaged over non-mosaics (Benda, 1969; Percy et al., 1993). However, detailed clinical assessment and long-term follow-up of these cases will be required for better understanding of this relationship and making use of these findings for counselling the affected patients especially mosaics. The results here also recommend the use of sensitive molecular techniques such as interphase FISH in clinical practice for diagnosis of chromosomal disorders, as FISH permits identification of the aneuploidy even when it is present in low grade (low level mosaicism) which may not be detected by using conventional karyotyping. This has particular relevance in terms of clinical management of the patients and counselling of the parents, especially for prenatal diagnosis in the next pregnancy.
Articles - Mosaicism in Down syndrome - D Modi et al.
In conclusion, the frequency of mosaicism as studied by FISH in DS individuals is greater (33%) than that reported previously (20%). However, since this frequency is still lower than that reported for trisomy 18 (100%) and Turner syndrome cases (75%), it is likely that different fetoprotective pathway(s) exist which facilitate the survival of DS individuals.
Acknowledgements This work was supported by a grant from the Department of Biotechnology, Ministry of Health and Sciences, India. We thank Dr Sudha G Gangal (Director) for her valuable suggestions. We are very grateful to the clinical staff of our Genetic clinic.
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