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650 JMed Genet 1995;32:650-653 Double non-disjunction in maternal II to a with 48,XXX, + 21 giving rise fetus J Med Genet: first published as 10.1136/jmg.32.8.650 on 1 August 1995. Downloaded from

Vicki M Park, Ralph R Bravo, Lee P Shulman

Abstract distributed throughout the genome, the se- We describe a prenatally detected case of gregation of any can be evaluated. double involving chromosome 21 The primary limitation of the molecular ap- and the (48,XXX, + 21) proach is that commonly used markers are along with determination of the se- located some distance away from the cen- gregation errors responsible for the double tromere, and integration ofthe into . The patient was ascertained the genetic map has not been achieved for most as a result of an abnormal maternal serum . Therefore, as with cytogenetic analyte screen showing an increased risk heteromorphisms, recombination events oc- for fetal Down's syndrome. Following de- curring between the polymorphism and the termination of the abnormal , centromere cannot be detected, potentially pregnancy termination was elected. Mi- leading to incorrect assessment of the stage of crosatellite polymorphisms and cyto- meiotic error. Studies comparing results from genetic heteromorphisms were used to cytogenetic heteromorphisms and from mo- determine that both arose as lecular markers have noted significant dis- a result of non-disjunction in maternal crepancies between the two systems with meiosis II. These results support hy- respect to stage of meiotic error.'31516 Such potheses that a segregation defect at a discrepancies could be explained by increased cellular level may cause non-disjunction recombination in pericentromeric regions.'718 involving more than one chromosome. However, there is no question that, when used judiciously, molecular approaches greatly im- (JtMed Genet 1995;32:650-653) prove our ability to study segregation. This is especially true ofthe type of case reported here, where segregation analyses of two chro- Although non-disjunction is the most common mosomes are required. cause of chromosomal abnormalities, the pres- ence two numerical abnormalities in a single of Case report conceptus is rarely observed. A number of such The parents were healthy and unrelated 19 cases have been reported,`7 but the mech- http://jmg.bmj.com/ year olds, and the mother had had one previous anisms by which double aneuploidies arise have pregnancy that resulted in the delivery of a not been well studied. We report a fetus with normal, healthy girl. In the current pregnancy, both trisomy 21 and in which both was performed at 17-5 weeks' aneuploidies arose by maternal non-disjunction following a "positive" serum analyte in meiosis II. gestation screen for fetal Down's (adjusted of chromo- syndrome Traditionally malsegregation risk of 1/120). Ultrasonography at the time of

somes has been studied using cytogenetic amniocentesis showed nuchal thickening on September 28, 2021 by guest. Protected copyright. This type of analysis is Department of heteromorphisms."9 (10 mm), a large ventricular septal defect, and relatively subjective, as visual differentiation of Obstetrics and a two vessel cord. The karyotype showed tri- Gynecology, subtle polymorphisms may be problematical. somy 21 and trisomy X in all 20 cells examined University of The usefulness of such analyses is limited to Tennessee, (fig 1 a). The parents elected pregnancy ter- those human chromosomes that frequently ex- Memphis, mination, which was performed at 19 weeks' 711 Jefferson Avenue, hibit heterochromatic polymorphisms, in- gestation. Routine confirmation of the fetal Room 429, Memphis, chromosomes and the cluding 1, 9, 16, karyotype could not be accomplished owing to TN 38163, USA acrocentric chromosomes. On the other hand, V M Park gross microbial contamination of all cultures R R Bravo* since the polymorphic heterochromatin is loc- of There- L P Shulman initiated from products conception. alised to pericentromeric regions, cytogenetic fore, interphase FISH (fluorescence in situ hy- themselves to the ana- Department of heteromorphisms lend bridisation) with probes for chromosomes X of a centromere driven event such as Pediatrics, lysis and 21 was used to confirm the presence of University of segregation. These studies have assumed an both in fetal tissue. The Tennessee, absence of recombination between the het- Memphis, of both parents were normal. USA eromorphism and the adjacent centromere. V M Park Available evidence from meiotic studies sup- *Present address: ports this assumptionl' but is limited to ob- Methods Servicio de Tocoginecologia, servations of normal male meioses. Cytogenetic analyses of GTG banded chro- Hospital Matemo- fluid and Infantil, San Luis, More recently, polymorphic DNA markers mosomes from amniotic peripheral Argentina. have been applied to the study of chromosome blood were performed according to standard Correspondence to: segregation."-4 Since DNA markers are highly procedures. Interphase FISH was performed Dr Park. polymorphic and very abundant, it is possible to according to a touch prep protocol from Image- Received 11 November 1994 in almost all families. netics, Inc (Framingham, MA). Briefly, a Revised version accepted for identify informative loci publication 2 March 1995 Furthermore, since DNA polymorphisms are freshly cut surface of fetal tissue was pressed Double non-disjunction in maternal meiosis II giving rise to a fetus with 48,XXX, + 21 651 a

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Figure 1 (a) Fetal katyotype from amniotic fluid, 48,XXX, + 21. (b) Partial katyotypes of chromosome 21 from the mother (left) and the father (right).

lightly against a clean microscope slide in order the remainder of the reagents in each tube. to deposit cells onto the slide. Following fixa- PCR conditions were 30 cycles of 940 (10 tion, slides were processed for FISH. The seconds), 550 (30 seconds), and 720 (30 sec- probes used to confirm the double trisomy were onds). The initial cycle was preceded by five a centromeric X chromosome probe (DXZ1) minutes at 940, and the final cycle was followed http://jmg.bmj.com/ and a cosmid probe from the long arm of by five minutes at 720. Reaction products were chromosome 21 (21 q22.3); hybridisation con- run on 6% polyacrylamide/urea gels followed ditions were those recommended by the sup- by autoradiography. For each locus examined, plier (Oncor, Inc, Gaithersburg, MD). Using observed alleles were assigned numbers based the DXZ1 probe, 98% of fetal cells exhibited on relative order of migration in the gel. three hybridisation signals, while control slides

containing normal diploid cells yielded three Results on September 28, 2021 by guest. Protected copyright. signals in only 0 5% of cells. Hybridisation of Microsatellite analysis was used to investigate the 21q probe was less efficient overall, but the parental origin of the extra chromosomes showed the presence of trisomy 21 cells in the observed in the fetus, and both the extra chro- fetal specimen. Approximately 10% of fetal mosome 21 and the extra X chromosome were cells exhibited three signals, while no such cells found to be ofmaternal origin (fig 2). Evidence were observed in control slides. of recombination was detected on both chro- DNA was extracted from either peripheral mosomes, since reduction to homozygosity was blood or minced fetal tissue by cell lysis, pro- observed at some alleles while maternal het- teinase K digestion, and phenol/chloroform ex- erozygosities were maintained at others. At the traction.19 Oligonucleotide primer sets specific more distal loci (D21 S 167 on chromosome 21 for microsatellite polymorphisms were ob- and MAOA, DXS556, and DXS538 on the X tained from Research Genetics (Huntsville, chromosome), the fetus had inherited three AL). Genotyping was performed by polymerase different alleles, two ofwhich were of maternal chain reaction (PCR) using a "hot start" pro- origin. Since the fetus had inherited two differ- tocol. AmpliWax beads were used according to ent maternal alleles at these loci, neither aneu- specifications provided by the supplier (Perkin could have arisen through postzygotic Elmer/Applied Biosystems). Briefly, each mitotic error. All other loci examined confirm 100 p1 reaction contained 10 mmol/l Tris-HCl, a maternal origin of the extra chromosomes. pH 8 3, 50mmol/1 KC1, 2 5 mmol/1 MgCl2, Based on visual inspection ofcomparative band 0-2 mmol/l each of dATP, dTTP, dCTP, and intensities within lanes containing fetal DNA, dGTP, 4 jCi 32P-dCTP, 0 5 mmol/l primers, 3 the fetus inherited a single maternal allele at units Taq DNA polymerase, and 50 ng genomic each proximal locus (D21S215, D21S258, DNA. Before initiation of the reaction, beads D21S236, D21S225, D21S11, and D21S222 were used to segregate enzyme and DNA from on chromosome 21 and AIAS2 and DXS981 652 Park, Bravo, Shulman Chromosome 21 - DNA marker studies D21S1 1 Mo Fe Fa Locus Mo Fe Fa J Med Genet: first published as 10.1136/jmg.32.8.650 on 1 August 1995. Downloaded from D21 S215 12 1 D21 S258 13 ... D21 S236 13 .; 2 D21 S225 12 D21 Sl 1 24 3 D21 S222 1 12 4 t - Crossover D21S167 X B0 23

X Chromosome - DNA marker studies Fetus = 233 Locus Mo Fe Fa DXS538 DXS981 DXS556 Mo Fe Fa MAOA 2 .4 Crossover ALAS2 . DXS981 12 M3 3 2 ~~~~~1 V. 3 Figure 2 Summary of informative microsatellite polymorphisms in the mother (Mo), fetus (Fe), andfather (Fa). Alleles shared by the mother and the fetus are highlighted by black boxes.

on the X chromosome). Each maternal allele Fetus = 113 is present at twice the dosage of the paternally Figure 3 Two loci showing maternal meiosis H non- derived allele (figs 2 and 3). disjunction of chromosome 21 (D21S11) and the X The loci closest to each centromere were chromosome (DXS981). Alleles observed in the mother

used to determine the stage of the maternal (Mo), fetus (Fe), andfather (Fa) are numbered http://jmg.bmj.com/ meiotic error. For chromosome 21, the relative sequentially. order of informative markers is according to McInnis et al.20 The most proximal locus is For the X chromosome, five informative loci D21S215, which is closely linked to cen- were identified (fig 2). Relative map order of tromeric DNA.2" In the fetus, all informative these loci is according to Schlessinger et al22 chromosome 21 markers except for D2 1S 167 and Brown et al.23 The single informative long ma- arm was DXS981, and the fetus inherited showed reduction to homozygosity of the locus on September 28, 2021 by guest. Protected copyright. ternally derived alleles (fig 2). For example, fig two copies of the maternal allele 1 (figs 2 3 (D21S11) illustrates double intensity of a and 3). Likewise, the most proximal short arm single maternal allele in fetal DNA. This is the locus, ALAS2, exhibited reduction to homo- expected pattern for non-disjunction occurring zygosity of maternal allele 2. These two mark- in the second meiotic division. For D21S 167, ers, flanking the centromere, indicate that the the fetus inherited both maternal alleles, 1 and X chromosome non-disjunction also occurred 4 (fig 2). Since D21S 167 was the most distal in maternal meiosis II. For the remaining three locus examined, this indicates the occurrence of informative markers, located distal to ALAS2 a crossover between D21S222 and D21S167. on Xp, heterozygosity ofthe maternally derived In addition to the molecular studies, G ban- alleles was maintained in the fetus. This in- ded metaphase preparations were evaluated for dicates the occurrence of a crossover between the presence of cytogenetic heteromorphisms ALAS2 and MAOA. involving chromosome 21. In the mother, there was a clear difference in the lengths of the stalk regions of the two copies of chromosome 21 Discussion (fig lb). This difference was not observed in We describe a case of prenatally detected metaphase preparations from the fetus, where double trisomy, in which both non-disjunction all three short arm segments appeared to be events occurred in maternal meiosis II. By approximately equal in length (fig la). This examination of microsatellite polymorphisms suggests that the fetus received a double dose on Xp, Xq, and 21 q, as well as cytogenetic of one maternal chromosome, rather than one heteromorphisms on 21p, we obtained con- copy ofeach homologue. As with the molecular sistent data from the pericentromeric region data, this observation is consistent with the of each chromosome arm. The observed 2lp occurrence of a meiosis II non-disjunction. heteromorphism would not have been in- Double non-disjunction in maternal meiosis II giving rise to a fetus with 48,XXX, + 21 653 1 Ford CE, Jones KW, Miller OJ, et al. The chromosomes formative on its own but, in combination with in a patient showing both mongolism and Klinefelter the molecular data, was useful in evaluating syndrome. Lancet 1959;i:709-10. 2 Hecht F, Nievaard JE, Duncanson N, et al. Double aneu- the stage of the meiotic error. As mentioned ploidy: the frequency of XXY in males with Down's

previously, recombination occurring between syndrome. Am J Hum Genet 1969;21:352-9. J Med Genet: first published as 10.1136/jmg.32.8.650 on 1 August 1995. Downloaded from 3 Mailhes JB, Moore CM, GershaniknI. A case of double the centromere and the most proximal marker trisomy in a liveborn infant: 48,XXY, + 13. Clin Genet can lead to incorrect assessment of the stage 1977;11: 147-50. 4 Townsend G. Dentition of a 48,XYY, + 21 male. Hum Genet of meiotic error. The use of pericentromeric 1982;61:267-8. loci on each chromosome arm minimises this 5 Therman E, Susman M. Human chromosomes: structure, be- havior, and effects. 3rd ed. New York: Springer-Verlag, possibility, since only a missed double crossover 1993:198-9. would lead to an incorrect conclusion. 6 Jaruratanasirikul S, Jinorose U. An infant with double tri- Double trisomies are rarely observed, pre- somy (48,XXX, + 18). Am JMed Genet 1994;49:207-10. 7 Tsukahara M, Fukuda M, Furukawa S. Double trisomy sumably because double non-disjunctions are (48,XXX, + 18). Am J Med Genet 1994;52:244. rare events, associated with inevitable lethality 8 Hassold TJ, Jacobs PA. Trisomy in man. Annu Rev Genet 1984;18:69-97. in most cases. Reported cases of multiple aneu- 9 Bricarelli FD, Pierluigi M, Landucci M, et al. Parental age have described liveborns exhibiting two and the origin of trisomy 21: a study of 302 families. Hum Genet 1989;82:20-6. viable aneuploidies (most often aneuploidy of 10 Laurie DA, HultenMA. Further studies on bivalent chiasma the sex chromosomes combined with either tri- frequency in human males with normal karyotypes. Ann Hum Genet 1985;49:189-201. somy 13, 18, or 21).'- The case described here 11 Antonaralis SE, The Collaborative Group. also involved a complement of this type (48, Parental origin of the extra chromosome in trisomy 21 as indicated by analysis of DNA polymorphisms. N Engl J XXX, + 21). Other cases ofmultiple aneuploidy Med 1991;324:872-6. involving at least one non-viable trisomy have 12 Sherman SL, Takaesu N, Freeman SB, et al. Trisomy 21: association between reduced recombination and non- been observed in spontaneous abortions.2425 disjunction. Am J7 Hum Genet 1991;49:608-20. Although few cases of multiple aneuploidy 13 Petersen MB, Frantzen M, Antonarakis SE, et al. Com- parative study of microsatellite and cytogenetic markers have been investigated (see below), several for detecting the origin of the nondisjoined chromosome mechanisms may be considered by which they 21 in Down syndrome. AmJHum Genet 1992;51:516-25. 14 Sherman SL, Petersen MB, Freeman SB, et al. Non-dis- might arise. All require a minimum of two junction of chromosome 21 in matemal meiosis I: evidence errors in . Random non-disjunction for a matemal age-dependent mechanism involving re- duced recombination. Hum Molec Genet 1994;3:1529-35. in both could lead to the formation 15 Stewart GD, Hassold TJ, Berg A, Watkins P, Tanzi R, of a doubly aneuploid zygote. Altematively, a Kurnit DM. Trisomy 21 (Down syndrome): studying global defect might cause multiple non- nondisjunction and meiotic recombination by using cyto- single genetic and molecular polymorphisms that span chro- disjunctions in the formation of a single . mosome 21. Am JHum Genet 1988;42:227-36. Available experimental evidence favours the 16 Lorber BJ, Grantham M, Peters J, Willard HF, Hassold TJ. Nondisjunction of chromosome 21: comparisons of second possibility as the predominant cause cytogenetic and molecular studies of the meiotic stage and of multiple aneuploidy. DNA parent of origin. AmJHum Genet 1992;51:1265-76. polymorphisms 17 Tanzi RE, Watkins PC, Stewart GD, Wexler NS, Gusella have been used to investigate parental origins JF, Haines JL. A genetic linkage map of human chro- of both aneuploid chromosomes in three cases mosome 21: analysis of recombination as a function of sex and age. Am J Hum Genet 1992;50:551-8. of spontaneous abortion, and in each case both 18 MacDonald M, Hassold T, Harvey J, Wang LH, Morton non-disjunctions occurred in a single parent.25 NE, Jacobs P. The origin of 47,XXY and 47,XXX aneu- ploidy: heterogeneous mechanisms and role of aberrant In these three cases, stage of meiotic error was recombination. Hum Molec Genet 1994;3:1365-71. http://jmg.bmj.com/ not determined for both aneuploidies. Parental 19 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual 2nd ed. New York: Cold Spring Harbor origin of the extra chromosomes has also been Laboratory Press, 1989. investigated in cases of sex chromosome poly- 20 McInnis MG, Chakravarti A, Blaschak J, et al. A linkage map of human chromosome 21: 43 PCR markers at somy. In reported cases, each aneuploidy res- average intervals of 2-5 cM. Genomics 1993;16:562-71. ults from segregation errors in a single 21 Warren AC, Petersen MB, Hul WV, et al. D21S215 is a (GT)8 polymorphic marker close to centromeric alphoid parent.2 128 For example, in reported cases of sequences on chromosome 21. Genomics 1992;13:1365-7. (49,XXXXX), there are four X 22 Schlessinger D, Mandel J-L, Monaco AP, Nelson DL, Wlllard HF. Report of the fourth intemational workshop on September 28, 2021 by guest. Protected copyright. chromosomes of maternal origin as a result of on human X chromosome mapping 1993. Cytogenet Cell successive X chromosome non-disjunction in Genet 1993;64: 147-94. 23 Browne DL, Luty JA, Litt M. Dinucleotide repeat poly- maternal meioses I and II.262829 It has been morphism at the DXS538 locus. Nucleic Acids Res 1991; postulated that the successive errors are related, 19:1161. 24 Borgaonkar DS. Chromosomal variation in man: a catalog of in that perturbed recombination may affect chromosomal variants and anomalies. 5th ed. New York: both meiotic segregations.'82526 In the case re- Alan R Iiss, 1989:689-90. the two non-disjunction events are 25 Zaragoza MV, Jacobs PA, James RS, Rogan P, Sherman S, ported here, Hassold T. Nondisjunction of human acrocentric chro- related by virtue of occurring not only in the mosomes: studies of 432 trisomic fetuses and liveborns. same parent, but in the same cell division. The Hum Genet 1994;94:411-17. 26 Hassold T, Pettay D, May K, Robinson A. Analysis of presence of a general cellular defect, such as nondisjunction in sex chromosome and pen- impaired spindle function or improper sig- tasomy. Hum Genet 1990;85:648-50. 27 David D, Marques RA, Carreiro MH, Moreira I, Boavida nalling of sister segregation, might MG. Parental origin of extra chromosomes in persons account for this type of event.3"33 with X chromosome tetrasomy. J Med Genet 1992;29: 595-6. Study of additional examples of multiple 28 Leal CA, Belmont JW, Nachtman R, Cantu JM, Medina aneuploidy are needed to determine the nature C. Parental origin of the extra chromosomes in X. Hum Genet 1994;94:423-6. of the errors in such cases. Evaluation of ex- 29 Martini G, Carillo G, Catizone F, Notarangelo A, Mingarelli ceptional instances of segregation failure un- R, Dallapiccola B. On the parental origin of the X's in a prenatally diagnosed 49,XXXXX syndrome. Prenat Diagn doubtedly will be useful in improving our 1993;13:763-6. understanding of the general mechanisms of 30 Lejeune J. Autosomal disorders. Pediatrics 1963;32:326-37. 31 Koshland D. : back to the basics. Cell 1994;77: non-disjunction. 951-4. 32 Carpenter ATC. Chiasma fimction. CeUl1994;77:959-72. We acknowledge the expert technical assistance of Ms Jody 33 Hawley RS, FrazierJA, Rasooly R. Commentary. Separation Lemer, and we thank Mr Russell Dodd for preparation of the anxiety: the etiology of nondisjunction in flies and people. figures. Hum Molec Genet 1994;3:1529-35.