Distribution of meiotic recombination along nondisjunction chromosomes 21 in Down syndrome determined using cytogenetics and RFLP haplotyping.
Hum Genet (1989) 83:280-286
© Springer-Verlag 1989
Distribution of meiotic recombination along nondisjunction chromosomes 21 in Down syndrome determined using cytogenetics and RFLP haplotyping Henk Meijer, Guus J. H. Hamers, Roselie J. E. Jongbloed, Gerrie P. M.Vaes-Peeters, Ren~ R.W.J. van der Hulst, and Joep P. M. Geraedts Department of Genetics and Cell Biology, Biomedical Center of the University of Limburg, Beeldsnijdersdreef 101, NL-6200 MD Maastricht, The Netherlands
Summary. Ten families (Down syndrome children and their parents) showing evidence of meiotic recombination between intraparental chromosomes transmitted after nondisjunction were studied. Cytogenetic polymorphisms and a cassette of RFLP markers distributed along chromosome 21 were used to analyze these families to localize the regions of meiotic recombination. Results indicated that only one crossover occurred per meiotic division and that nine of ten nondisjunctions appeared to be of maternal origin. In one family the crossover had taken place in the pericentromeric region, proximal to marker D21S13, which is quite exceptional A chance of meiotic recombination within region 21q21, flanked by marker D21S72 and the amyloid gene, could be demonstrated in seven of the ten families. Most strikingly, this chance significantly decreased distal to q21, with frequencies of 0.3 and 0.1 in regions q22.2 and q22.3-qter, respectively. It is hypothesized that decreased chiasmata formation in the most distal part of chromosome 21q might promote nondisjunction. Furthermore, data from the ten crossovers made it possible to map provisionally two previously undefined markers, D21S24 and D21S82, to regions q21-qter and q22.1-qter, respectively.
Introduction In recent years cytogenetic and molecular genetic studies on chromosome 21 have been designed to elucidate the molecular basis of genetic defects leading to several clinical pictures. Down dyndrome (DS, trisomy 21) is the most common cause of mental retardation, afflicting about i in 700 liveborn infants (Adams et al 1981). The condition is associated with an increased risk of acute leukemia (Scholl et al. 1982) and features of premature aging (Martin 1978) and dementia. "Regular" trisomy 21 occurs in roughly 95% of cases of Down syndrome and is the result of chromosomal nondisjunction in one of the parents. Compiled studies of Juberg and Mowrey (1983) showed that 80% of DS cases result from a nondisjunction of maternal origin. The percentage meiotic errors occuring during meiosis I is 80% in mothers, but 60% in fathers. It is generally accepted
Offprint requests to: H. Meijer
that increased maternal age is associated with an increased risk for DS offspring (Smith and Warren 1985), but the underlying genetic defects are the subject of various conflicting hypotheses (Mattei et al. 1980). The contribution of paternal meiotic errors to DS is also open to discussion (Mikkelsen 1982). Currently the interest in the pathogenesis of DS has been greatly stimulated by several studies suggesting an association of at least one type of Alzheimer's disease (AD), the most common type of presenile dementia, and a chromosome 21 defect (Goldgaber et al. 1987; Tanzi et al. 1988; St GeorgeHyslop et al. 1987). Alzheimer's disease and Down syndrome share some patholoigcal features including premature aging and dementia. In the brains of both types of patients an accumulation of amyloid protein, causing typical neuropathological changes, is seen (Wisniewsky et al. 1985). Intriguing hypotheses have been proposed concerning a possible genetic predisposition as a major promoting factor causing meiotic nondisjunction of chromosome 21 (Antonarakis et al. 1985). Such a feature might be revealed by altered (reduced?) meiotic recombination as has been suggested by Warren et al. (1987) and Antonarakis et al. (1986). To test the above-mentioned hypotheses, we studied the distribution of meiotic recombination occurring along nondisjunction chromosomes 21. Using cytogenetic polymorphisms and a cassette of RFLP markers distributed along chromosome 21, we analyzed in detail ten families (DS child and its parents) showing evidence of meiotic recombination between the parental chromosomes transmitted together after nondisjunction.
Materials and methods
Selection of families with regular trisomy 21 offspring Thirty-three families with trisomy 21 offspring, cytogenetically established either postnatally or prenatally, were selected for a large multidisciplinary study on the cause of Down syndrome. All were isolated "regular" cases of trisomy 21, and no other chromosomal abnormalities were detectable. Based on molecular analysis with a set of RFLP markers described below, ten DS families showed evidence of meiotic recombination and were selected for detailed analysis. Nine
281 nondisjunctions were of maternal origin (mean age 31 years), and one was of paternal origin (age 29 years).
Cytogenetic analysis Lymphocyte cultures obtained from blood samples were used for karyotype analysis. Chromosome slides were Q-banded with Atabrine to study the fluorescence polymorphisms in the short-arm region of chromosome 21 (Hamers et al. 1987). At least ten well-spread metaphases of each individual were examined to compare the intrafamilial short arm (21p) variants to determine the parental origin and the meiotic stage (error) leading to nondisjunction 21.
Molecular analysis High molecular weight DNAs were isolated from the leukocyte fractions blood samples of each individual (Weening et al. 1974), and purified leukocyte D N A (10 gg) was digested to completion with a twofold excess of the appropriate restriction endonuclease (PstI, HindIII, BglII, TaqI, EcoRI, XbaI, MspI, or BamHI) according to the manufacturer's instructions (Boehringer, Mannheim). D N A fragments were separated by 0.7%-0.9% agarose gel electrophoresis and transferred to Gene Screen Plus membranes (New England Nuclear) according to Southern (1975) as slightly modified by New England Nuclear. Hybridization conditions and autoradiographic pro-
Table 1. Core set of polymorphic DNA markers on chromosome 21 with well-defined map positions
Map position
Locus
Clone designation
Restriction enzyme
Allele size (kb)
References
21q11.1
D21S13
G21RK
TaqI
Stewart et al. (1985a, 1988)
21qll.l-q21
D21S16
pGSE9
XbaI
21q11.2-q21
D21S72
pG95-1i
TaqI
A1 A2 A1 A2 A1 A2 B1 B2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 B1 B2 A1 A2
EcoRI 21q21-q22.1
Amyloid
9-110
BglII
21q22.1
SODI
pS61-10
BglII
21q22.2-q22.3
D21S17
pGSH8
BglII
21q22.3
D21S15
pGSE8
MspI
21q22.3
D21S25
10.2
HindlII
21q22.3-qter
D21S19
pGSB3
PstI
21q22.3-qter
BCEI
pS2
BamHI
7 6 7.3 6.4 3.95 5.55 1.95 2.95 9.6 6.9 5.1 3.6, 1.5 18.5 12.3 4.1 3.4 9.5 8.5 3.6 2.2 1.6 1.5 8.5 3.5
Stewart et al. (1985a, 1988) Goodfellowet al. (1986) Tanzi et al. (1988) Rudd et al. (1988) Blanquet et al. (1987) Van Broeckhoeven et al. (1987) Tanzi et al. (1988) Kittur et al. (1985) Tanzi et al. (1988) Stewart et al. (1985a) Tanzi et al. (1988) Stewart et al. (1985a) Millington-Ward et al. (1985) Mtinke et al. (1988) Stewart et al. (1985b) Tanzi et al. (1988)
Moisan et al. (1985)
Table 2. Polymorphic DNA markers on chromosome 21 with less defined map positions
Map position
Locus
Clone designation
Restriction enzyme
Allele sizes (kb)
References
21
D21S82
Fr8-77
BamHI
A1 A2 A3 B1 B2 B3 A1 A2 B1 B2 B3 A1 A2 B1 B2 A1 A2
Xiao et al. (1987)
EcoRI 21pter-q21.1
D21S26
26C
PstI
21q21.2-qter
D21S42
SF43
TaqI PstI
21p
D21S24
p21.3
PstI
4.5 4.3 4.0 4.2 4.0 3.7 3.0 2.5 16.5 14.0 4.8 3.9 2.4 1.6 0.8 4.0 3.5
Millington-Ward et al. (1985)
Korenberg et al. (1987)
Millington-Ward et al. (1985)
282 cedures have been previously described (Hamers et al. 1987). D N A probes were radiolabeled with ct32p-dCTP by means of the random primer method essentially as described by Feinberg and Vogelstein (1983), the chemicals and protocol being provided by Boehringer Mannheim. The following probes, inserts isolated from recombinant plasmids or bacteriophages, were used: D21S24, D21S25, D21S26, and D21S42 (kindly provided by Dr.A.Milington Ward, University of Leiden, The Netherlands; pS61-10 (SODI), pS2 (BCEI), and G21RK (D21S13) (purchased from the American Type Culture Cellection, ATCC; and D21S72 (pG95 c~-l-lla) (a generous gift of Dr. B. N. White, Queens University of Kingston, UK). Clone Fr8-77 (D21S82) was a kind gift of Dr. G. Scherer, Institute of Human Genetics, Freiburg, FRG. Probes D21S15, D21S16, D21S17, and D21S19 were kindly supplied by Dr. G. D. Stewart, Howard Hughes Medical Indstitute, Michigan, USA. The probe amyloid c-DNA (clone 9-110) was kindly provided by Dr.Jie Kang, Institut ftir Genetik, K61n, FRG. From this clone, a 745-bp TaqI fragment (spanning base pairs 1730-2475) was isolated, which detects two allelic BglII fragments (Kang et al. 1987; van Broeckhoven et al. 1987). Tables 1 and 2 summarize the relevant data of the clones used, including allelic sizes of the polymorphic restriction fragments and the subregional map positions of the markers according to the most recently published data.
Data analysis With use of the pericentromeric cytogenetic markers or each of the RFLP markers described above, it was possible to distinguish between the following possibilities: uninformative; meiosis I (either parent); meiosis II (either parent); maternal (meiotic stage unknown); paternal (meiotic stage unknown); maternal meiosis I; maternal meiosis II; paternal meiosis I; paternal meiosis II; maternal I excluded; maternal II excluded; paternal I excluded; paternal II excluded; if maternal, then meiosis I; if maternal, then meiosis II; if paternal, then meiosis I; if paternal, then meiosis II. Initially the eytogenetic markers and a core set of RFLP markers for which the precise map positions and linkage order had been deduced from published data were selected from Table 1. These were (from proximal to distal 21q) D21S13D21S16- D21S72-Amyloid- SODI-D21S17-D21S15-D21S19. The procedure described by Stewart et al. (1988) was used to order data in a way for easy establishment of the area in which crossing-over had taken place. Next, the data from the remaining markers, D21S25 and BCEI (from Table 1) and D21S26, D21S82, D21S42, and D21S24 (from Table 2), were entered with the aim to map them between the other markers, based on the assumption of the least necessary crossing-overs.
Results
Figure 1 gives the results of cytogenetic and RFLP typing of the chromosomes 21 in ten families (DS children and their parents) showing evidence of meiotic recombination between the parental chromosomes transmitted after nondisjunction. The set of RFLP markers is shown in each figure according to their most recently published map positions. The data of the other RFLPs are shown below each family set.
In family I the cytogenetic markers were compatible with a maternal meiosis I error, whereas D21S17 and SODI indicated a meiosis II origin. Since the intermediate markers were not informative, it can be concluded that meiotic recombination must have taken place between the cytogenetic markers and SODI. In family II the cytogenetic markers indicated a maternal meiosis I error. Maternal origin was confirmed by markers Amyloid and D21S17. In addition Amyloid pointed toward a meiosis II error, as did D21S24, suggesting that a meiotic recombination must have taken place between the cytogenetic markers and the amyloid gene. The fact that D21S26 was informative for a meiosis I pattern suggests that this marker is proximal to the amyloid gene and D21S24, although its exact map position could not be established. In family III the cytogenetic markers and both D21S13 and D21S72 indicated a maternal meiosis I nondisjunction, whereas D21S17 suggested a meiosis II error. Since the adjacent markers Amyloid and SODI were not informative, it could be concluded that the crossover occurred somewhere between D21S72 and D21S17. The data obtained from the other markers strongly suggest that D21S26 is located proximal to D21S17 and that D21S24 is situated distal to D21S72. The data also confirm that D21S42 and D21S25 are localized distal to D21S72. In family IV the maternal origin of the meiotic error was indicated by the cytogenetic markers as well as by markers D21S24 and D21S26. Therefore D21S13 indicated a meiosis I nondisjunction. However, since SODI and D21S17 showed a meiosis II pattern, it could be concluded that meiotic recombination had occurred in an area between D21S13 and SODI. The remaining informative probe D21S24 also indicated a meiosis II pattern, strongly suggesting its location to be distal to D21S13. The cytogenetic markers in family V indicated a meiosis II error of paternal origin, the meiotic stage being confirmed by markers D21S72. However, the remaining informative markers (Amyloid, D21S19, D21S24, and D21S25) proved a meiosis I error, providing evidence that meiotic recombination had taken place in an area between D21S72 and Amyloid. Furthermore the data confirmed that D21S24 and D21S25 must be located distal to D21S72. In family VI the maternal origin of the meiotic error was proved by D21S25 and D21S26. Taking this into account, the cytogenetic markers revealed a meiosis I error, which was confirmed by RFLP markers Amyloid and SODI. Marker D21S82, however, proved a meiosis II nondisjunction, giving rise to the conclusion that a crossover occurred distal to the SODI locus and strongly suggesting that D21S82 must be located distal to SODI. In addition, D21S24 proved a meiosis I error, suggesting that this marker is situated proximal to D21S82. In family VII only the cytogenetic markers indicated a meiosis I pattern of maternal origin. Even the most proximal marker, D21S13, indicated a meiosis II error, which was confirmed by D21S72 and the Amyloid marker. Unfortunately all of the markers distal to the amyloid gene were not informative, but nevertheless the results provide evidence for at least one crossover in the pericentromeric region, proximal to D21S13. In family VIII both the cytogenetic markers and D21S16 showed a meiosis I pattern of maternal origin. In contrast D21S17 and D21S19 indicated a meiosis II error, providing
283
,21 marker Father IMothe, Child
T 21 marker Father Mothel Child meiotic error
13 ~ C Y t o
ab
cd
acd
mat MI
13 ~ C Y t O
ab
cd
acd
meiotic error
1
ltl
11.2
mm
D21~2
12 D2181~ 22 )21S72 12
ii 22 12
iii 222 112
pat MI excl not inf. not inf.
~r
~kmylok 12 ii 50DI
12 12
112 iii
not inf. • if m a t :MII
~17
ii 12 22
12 12 22
ill 122 222
if mat ,MII not inf. not inf.
Ii ii ii ii
ii 22 ii 22 ii 22
iii 122 Iii 122 Iii 222
~ot inf. naternal not inf. hate ~nal 0at HI excl not inf.
2,%1
= j
)21S15
22.3
)21S19
21
~s2= )21826 )2182~ )21S8; 321S4; ~]CE I
12 22
- - ~ / 11.1 /~D21S13
12
ii
22
iii 122 222
pat MI excl pat M I I e x c l pat MI excl
)21S72 12
iI Ii
12 ii
122 iii
m a t MII j K not inf.
)21S17 22 )21815 12 )21S19 22
Ii 12 22
112 122 222
maternal not inf. not inf.
D21S24 12
if mat:MII not inf. if m a t : M I pat MI exc] not inf, not inf.
@2181~ 12
11.2
22
I
11.1 11,1 ~ 11 2
13 ~
Cyto
ab
ce
bcc
~aternal
22.2 =
22.3 21
12
iii
D21S2~:
22
222
]CEI
13 22 22 22
113 222 222 222
12 D21S2(~ ii ID2188~ 12 !D21S4~ 22
22
D 2 1 ~ 22 D21SM 12
Cyto
13
12
1~2 o o t ~
ii
ii
iii
not inf.
D21S17 Ii D21S15 ii
12 12 ll
iii :122 ii12
if m a t : M I ~ ~at MII pat MII ex¢
12 12 13
L22
22
[33 222
12 22
LII 222
mat MII if m a t : M I I if m a t : M I pat MIexel. if m a t : M I I not inf.
ab
. ~s,9
21
cd
12
D21S2dii
D ~ $ 2 5 12 D21S26 33 D21S82 12 D21S42 12
BCEI
22
~22
F a m i l y VI
bbe
T21 t
marker Father Mothe 3hild meiotic error
ic error
pat
13 ~ C y t o
MII
ab
bc
bbc
i£ mat:Ml
D21S13 ii D21S16 12 )21S72 12
ii 22 22
iii 122 122
not inf. pat MII exc pat MII exc
~mylok ii 30DI ii
12 12
112 112
if mat:MI if mat :MI
)21S17 12 )21S15 12
22 12
122 122
~S19 22
22
222
pat MII exc not inf, not inf.
ID21S24 ii
12 Ii 22 12 12 22
112 112 122 222 112 222
if mat:MZ maternal maternal if mat:MiI not inf. not inf.
p12
--"
~2,~
I1.1 11.2
D21S7211 2
12 12 12
122 122 122
if mat::MI not inf. -Ir not inf,
~ylo~ 12 SODI ii
ii 12
lll iii
pat MI e x ~ if mat:Ml
Amylo~dl2 SODI l !
~1S17 1 2 321S15 ii )21S19 22
12 ii 22
ii iii
if m~at:MII not inf. not inf.
321S17 12 321S15 ii )21S19 12
321S24122 :)21S25112 ~21826122 )21S82 ~3
12
112
D21S1S 22 D21S72 12
ll
2.
22
222
±i"
112
22
222
~-~~
~ot ~ f .
q
ll
111
if pat:MI not inf.
22.1 22.2
22.3
21
D21S42
~CEI
a
22
222
22
222
ii 22 22 22
112 222 222 222
2;
~at MII pat MI excl ~aternal pat MI excl not inf. not inf.
aa
bc
~
o2,s2,
D21S25 D21S26 ~2 D21SB2 D21S42 ~ BCEI 22
Famil
F a m i l y VII
abe
mat MI
12 22
1!2 112
22
122
~
,22
~ ~a~:.~
112 222 223 222
if pat:MI not inf paternal mat MI excl
22 13 12
22
222
marker Father
not inf. ~aternal if pat:MI
not inf,
MothelChild
mm
2Zl
m
22.2 r o l e
22-3 21
D21S25 22 D21S26 ii D21S82 22
D21S42 12 BCEI 22
~eiotic error
ad
bc
abe
sat MI
D21S13 12 )21816 22
12 12 22
112 122 122
lot inf. Lf mat:MI ~at MII ex~
Amylok 12 SODI
12
ii ii
iii 112
)at MI excl ~at MIIexcl
~i~17 121S15 D~sm
22 i2 ii
12 12 12
222 if m a t : M I ~ 112 not inf. 122 nat MII
DinS24 12 12
12 22
122 not inf. -222 not inf. 222 pat MI excl 222 not inf. 222 not inf. 122 pat MII axe
13 ~ C Y t o
2~
Vlll T21
T21 marker ;Father MotherChild meiotic error 13 ~ C Y t o
11.1 11.2
not inf. if' pat :MII
21
q 21
p12
p12
~
11.2
~
11.~
11.1
D21S13 12
mm
22 22
D2181E )21S7~
12 22 12
iii 222 112
if m a t : M I I ; n o t inf. mat MII
m
~mylok 12 ii ~ODI
12 ii
I11 iii
22.2 m
21
m
)215"17
ii 12 ii
iii 122 iii
not inf. not inf. pat PI exc
Ii 12 11 22 22 22
112 }22 iii 223 222 222
pat M I I e x c not fnf. pat MI exc patMIIexe not inf. pat MI exc
ii
)21S15 12 )21S19 12
~s24
D21S2E
12 12
D21S82 D21S4~ BC E I
23 22 12
D21S2e 12
/
if mat:MII not inf,
D21S72 12
2Zl
22.1
22-3
11.1 11.2
q 21 m
q 21 m
22.?. m
2z3 21
D21825 D21S26 D21S82 D21S42 BCE I
IT21 marker Father Molhe 2hild meiotic error 13 ~ C y t o
ab
cd
12
22 22 12
22 22 22 22
Family X
F a m i l y IX
acd
mat MI
1,211
marker Father MotherChild 13 ~ C y I o
ab
meiotic error
ad
acd
~at MI
22 22 22
-~22 222 L22
pat PI excl not inf. pat PII exc]
ii ii
[Ii [Ii
not inf. not inf.
lD21817 12 ;D21S15
ii
iii
D21S19
22
12
[12
pat PI exel not done A mat MII
D21S24 12 D21S25 ii D21S26 12
12 22 ii
L22 not inf. 1-22 maternal 112 pat PII e x c
22
222 222
p12
p12 11,2
1%1 11.1 ~ 11.2
mB
D21513 Ii D21816 22 ID21872 12
22 22 ~i
122 222 132
maternal Aot inf. pat M I I ex(
Amylok 12 SODI 11
12 12
122 112
not inf. if m a t : M Z
m
11.2 11.1 - - 11.1 ~ D 2 1 S 1 3 12 D21816 22 11.2 q 21
q 2~ m 22.1
m
)21S72
12
~,mylok II ~ODI
ii
2~ m
22_3
21 b
if m a t : M l not inf. if m a t : M I
11.2
11.1 _ _ 11.2
22.2
112 222 112
V
marker Father Mother Child mei
p12
22.~ m
22.3L.
11._~1 .
11,2
12 22 12
D21S72 Ii
I _ _ ~,?~ 12
11.2
11.2
321S13 ii
,D21S16 22
m
22.1
~eioti¢ error
p 12
if mat :MI
211BB m~mylok ~ODI
Famil T21
aab
t
• 11.2
Family IV marker Father MotherChlld
ab
p12
q 21
. 2~ m m
~etotic error
aa
13 ~ C Y t o
I1.2
"-_____L~
T21 ~otherChild
marker Father
m a t MI
p12
p12 11.2 ~
22.2
Family III
Family II
Family I.
D21S17 ii D21S15 22 D21S19 II
12 ii 12
112 112 IIi
ii 22 Ii
22 12 ii
122 -222 IIi
22 22
22 22
o~s24 D21S25 D'ZIS26 D21S82 D21S42 BC E I
23
22
mat MI matern81~[ if m a t : M fr
maternal if m a t : M I I not inf. 222 p a t M I exol 222 not 5nf. 222 not inf.
22.2
2Z.3
m 2]
D21S82 12 D21S42
BCEI
22 22
22 22
222
pat ~I excl not inf. not inf.
Fig. l a , b. Cytogenetic and RFLP typing of chromosomes 21 of ten D o w n syndrome children and their parents. A core set of markers is shown according to their published map position and mutual orientation. The remaining markers are shown below each family set
284 0 marker r 13
p12
3. Region q22.1 including SODI
~Cyto
4. Region q22.2 including D21S17 and D21S15 5. Region q22.3 including D21S19 Subsequently each of the crossovers were assigned to one or more of those five regions. The accumulated data as presented in Fig. 2 show the chance of a crossover event occurring in a particular chromosomal region.
1
Discussion
F21
Fig. 2. Subregional distribution of the chances of meiotic recombination along nondisjunction chromosomes 21 of ten Down syndrome children
13 ~ C y t o -'----
p12
q
)21S24
Fig. 3. Chromosome 21 subregional map positions of RFLP markers D21S24, D21S25, D21S26, D21S42, D21S82, and BCEI related to the core RFLP marker set
evidence of a meiotic recombination event between D21S16 and D21S17. Since both SODI and Amyloid were not informative, the crossover area could not be defined further. In family IX the cytogenetic markers and both markers SODI and D21S17 demonstrated a maternal meiosis I stage whereas, in contrast, the most distal marker D21S19 indicated a meiosis II error, indicating that a crossover must have taken place between D21S17 and D21S19. In addition, D21S25 proved a meiosis II error, strongly suggesting this marker to be situated distal to D21S17 In family X the cytogenetic markers indicated a meiosis I error of maternal origin, whereas D21S19 provided evidence for a maternal meiosis II error. Except D21S25, confirming only the maternal origin of the meiotic nondisjunction, none of the other markers were informative. Meiotic recombination could have happened anywhere on chromosome 21 except distal to D21S19. To obtain a clear picture of the distribution and preference of the crossover regions of the different nondisjunction chromosomes 21, we processed the data from Fig. 1 as follows. First, five crossover regions covering the whole chromosome 21 were defined as follows: 1. The satellite and pericentromeric area enclosed by the cytogenetic markers and the RFLP couple D21S13 and D21S16 2. Region q21 enclosed by D21S72 and Amyloid
With pericentromeric cytogenetic markers and a core set of eight RFLP markers distributed along the long arm of chromosome 21 it was possible to trace and define the area enclosing crossover events along nondisjunction chromosomes 21 of ten Down syndrome children. Based on testicular biopsy studies of normal males, a mean chiasmata frequency per meiotic division of about 1 for chromosome 21 was assumed (Lauri and Hult6n 1985). One might thus argue that multiple crossovers in chromosome 21 will not readily occur, and indeed, using the cytogenetic markers and the core set of RFLP markers, none of our recombinant families showed evidence of more than one crossover event per chromosome 21. Taking this assumption into account, with the recombination data of families II, III, IV, V, VI, and IX we were able to map provisionally some poorly assigned markers (indicated in Table 2 and below each family set in Fig. 1). The data from family VI strongly indicated that the D21S82 locus is situated distal to the SODI gene, somewhere within region q22.1-qter, and also suggested that D21S24 is located proximal to D21S82. On the other hand, according to the data from family III, D21S24 should be placed distal to the D21S72 locus. Combined, these data suggest that the position of D21S24 is somewhere within region q21-qter, which completely differs from its previously published position on 21p (Milington-Ward et al. 1985). In addition, data from family II strongly suggested that D21S24 is located distal to D21S26 and furthermore confirmed the previously published position of D21S26 within region pter-q21.1. Taken together, the relative orientation of these three loci from proximal to distal probably is D21S26, D21S24, D21S82, although their exact map positions remained obscure. The localization of D21S25 is well known (Mtinke et al. 1988), and the results obtained from family IX confirm this position and also strongly suggest that this marker is located distal to D21S17. Since D21S15 was not informative, the exact linkage order could not be established. The two possibilities are D21S17-D21S25-D21S15 and D21S17-D21S15-D21S25. The same holds true for the most distal markers, D21S19 and BCEI, giving rise to the possibilities D21S15-D21S19-BCEI and D21S15-BCEI-D21S19. Mapping data of the markers discussed above, together with the core set of RFLP markers, are summarized in Fig. 3. The distribution of crossover events along the nondisjunction chromosomes 21 gives rise to the following comments: the processed data from Fig. 1 as shown in Fig. 2 suggest a moderate chance of meiotic recombination in the satellitepericentromeric region. A t least one of ten families (family VII) clearly shows a crossover in this region. Moreover in three other families (I, II, and X) the crossover might have also occurred in this region. Reduced meiotic recombination
285 within the pericentromeric region of chromosome 21 has been demonstrated (Kurnit 1979; Stewart et al. 1988; Warren et al. 1987). It has been suggested that in nondisjunction chromosomes 21, the chance of meiotic recombination may be very low in this region, although our data clearly do not support such a theory. We have demonstrated that in seven of ten DS families the crossovers could have occurred in region q21, whereas the chance of meiotic recombination in the area distal to q21 (particularly within regions q22.2 and q22.3) has been found to be markedly lower. The latter observation is quite surprising because it suggests decreased chiasmata formation in the most distal part of 21q, a phenomenon not known to occur in normal functioning chromosomes. So a tempting possibility might be that this is a genetic feature (defect?) associated with (or predisposing to) nondisjunction of chromosomes 21. Here we meet again the hypothesis of reduced meiotic recombination as a possible cause of nondisjunction of chromosome 21, as recently proposed by Antonarakis et al. (1986) and Warren et al. (1987). In the studies of Warren et al. (1987) the hypothesis was tested by comparing linkage maps of chromosomes 21 that had disjoined normally and chromosomes 21 that had undergone nondisjunction. However the evidence for their conclusion is not very convincing because the authors did not provide clear insight into the real number of haplotyped recombinant nondisjunction chromosomes 21. Moreover, by using rather few RFLP markers, one could have easily underestimated possible crossovers. Hamers et al. (submitted for publication), using a more direct approach, have analyzed in detail the rate of recombination of chromosomes 21 involved in nondisjunction. Their and our studies presented here lead us to suggest that the hypothesis of reduced meiotic recombination as a cause of nondisjunction, if true, will be probably be restricted to a limited distal area of chromosome 21q. Sound proof to support this theory should come from future studies that analyze many more DS and normal families and, above all, include many more RFLP markers equally covering chromosome 21.
Acknowledgements. The authors wish to thank Drs. A.MillingtonWard, B.N. White, G. Scherer, G.D. Stewart, and J. Kang for their kind gifts of the various probes and their interest in our studies. Dr. J.P. Fryns is thanked for critically reading the manuscript.
References Adams MM, Erickson JD, Layde PM, Oakley GP (1981) Down syndrome: recent trends in the United States. JAMA 246:758-760 Antonarakis SE, Kittur SD, Metaxotou C, Watkins PC, Patel AS (1985) Analysis of DNA haplotypes suggests a genetic predisposition to trisomy 21 associated with DNA sequences on chromosome 21. Proc Natl Acad Sci USA 82: 3360-3364 Antonarakis SE, Chakravarti A, Warren AC, Slangenhaupt SA, Wong C, Halloran SA, Metaxotou C (1986) Reduced recombination rate in chromosomes 21 that have undergone nondisjunction. Cold Spring Harbor Symp Quant Biol 51 : 185-190 Blanquet V, Goldgaber D, Turleau C, Creau-Goldberg N, Delabar J, Sinet PM, Roudier M, Grouchy J de (1987) The [~-amyloidprotein (AD-AP) cDNA hybridizes in normal and Alzheimer individuals near the interface of 21@1 and q22.1. Ann G6n6t (Paris) 30: 6869 Broeckhoven C van, Genthe AM, Vandenberghe A, Horsthemke B, Backhovens H, Raeymakers P, Van Hul W, Wehnert A, Gheuens J, Crast P, Brnyland M, Martin JJ, Salbaum M, Multhanp G,
Masters CL, Beyreuther K, Gurling HMD, Mullan MJ, Holland A, Barton A, Irving N, Williamson R, Richards SJ, Hardy JA (1987) Failure of familial Alzheimer's disease to segregate with the A4-amyloid gene in several European families. Nature 329: 153-155 Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132 : 6-13 Goldgaber D, Lerman MI, McBride OW, Saffiotti U, Gajdusek DC (1987) Characterization and chromosomal localization of a c-DNA encoding brain amyloid of Alzheimers disease. Science 235 : 877880 Goodfellow PJ, Duncan AMV, Simpson NE, White BN (1986) Three RFLPs recognized by an anonymous sequence localized to 21q11.2 [HMG8 D21S72]. Nucleic Acids Res 14 : 4375 Hamers AJH, Vaes-Peeters GPM, Jonngbloed RJE, Millington-Ward AM, Meijer H, Die-Smulders CEM de, Geraedts JPM (1987) On the origin of recurrent trisomy 21: determination using chromosomal and DNA polymorphisms. Clin Genet 32 : 409-413 Juberg RC, Mowrey PN (1983) Origin of non-disjunction in trisomy 21 syndrome; all studies compiled, parental age analysis and international comparisons. Am J Med Genet 16:111-116 Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik K-H, Multhaup G, Beyreuther K, Muller-Hill B (1987) The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325 : 733-736 Kittur SD, Antonarakis SE, Tanzi RE, Meijers DA, Chakravarti A, Groner Y, Philips JA, Watkins PC, Gusella JF, Kazazian Jr HH (1985) A linkage map of three anonymous human DNA fragments and SODI on chromosome 21. EMBO J 4 : 2257-2260 Korenberg JR, Croyle ML, Cox DR (1987) Isolation and regional mapping of DNA sequences unique to human chromosome 21. Am J Hum Genet 41 : 963-978 Kurnit DM (1979) Satellite DNA and heterochromatin variants: the case for unequal mitotic crossing over. Hum Genet 47 : 169-186 Lauri DA, Hultdn MA (1985) Further studies on bivalent chiasmata frequency in human males with normal karyotypes. Ann Hum Genet 49:189-201 Martin G (1978) Genetic syndrome in man potential relevance to the pathobiology of aging. Birth Defects 14: 5-39 Mattei JF, Ayme S, Mattei MG, Giraud F (1980) Maternal age and origin of non-disjunction in trisomy 21. J Med Genet 17 : 368-372 Mikkelsen M (1982) Parental origin of the extra chromosome in Down's syndrome. J Ment Defic Res 26 : 143-151 Millington-Ward A, Wassenaar ALM, Pearson PL (1985) Restriction fragment length polymorphic probes in the analysis of Down's syndrome. Cytogenet Cell Genet 40:699 Moisan JP, Mattei MG, Baeteman-Volkel MA, Mattei JF, Brown AMC, Garnier JM, Jeltsch JM, Masiakowsky P, Roberts M, Mandel JL (1985) A gene expressed in human mammary tumor cells under estrogen control (BCEI) is located in 21q22.3 and defines an RFLP. Cytogenet Cell Genet 40:701-702 Mfinke M, Foellmer B, Watkins P, Cowan J, Caroll AJ, Gusella JA, Francke U (1988) Regional assignment of six polymorphic DNA sequences on chromosome 21 by in situ hybridization to normal and rearranged chromosomes. Am J Hum Genet 42 : 542-549 Rudd NL, Dimnik LS, Greentree C, Mendes-Crabb K, Hoar DI (1988) The use of DNA probes to establish parental origin in Down syndrome. Hum Genet 78:175-178 Scholl T, Stein Z, Hansen H (1982) Leukemia and other cancers, anomalies and infections as causes of death in Down's syndrome in the United States during 1976. Dev Med Child Neuro124: 817829 Smith GF, Warren ST (1985) The biology of Down syndrome. Ann NY Acad Sci 450 : 1-9 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bio198:503-517 Stewart GD, Harris P, Gait J, Ferguson-Smith MA (1985a) Cloned DNA probes regionally mapped to human chromosome 21 and their use in determining the origin of non-disjunction. Nucleic Acids Res 13 : 4125-4135 Stewart GD, Tanzi RE, Gusella JP (1985b) I@LPs at the D21S19 locus of human chromosome 21. Nucleic Acids Res 13 : 7168
286 Stewart GD, Hassold TJ, Berg A, Watkins P, Tanzi R, Kurnit DM (1988) Trisomy 21 (Down syndrome): studying non-disjunction and meiotic recombination by using cytogenetic and molecular polymorphisms that span chromosome 21. Am J Hum Genet 42 : 227-236 St George-Hyslop PH, Tanzi RE, Polinsky R J, Haines JL, Sorbi S, Piacentini S, Stewart GD, Hobbs WJ, Conneally PM, Gusella JF (1987) The genetic defect causing familial Alzheimer's disease maps on chromosome 21. Science 235 : 885-890 Tanzi RE, Haines JL, Watkins PC, Stewart GD, Wallace MR, Hallewell R, Wong C, Wexler NS, Coneally M, Gusella JF (1988) Genetic linkage map of human chromosome 21. Genomics 3 : 129136 Warren AC, Chakravarti A, Wong C, Slangenhaupt SA, Halloran SL, Watkins PC, Metaxotou C, Anatonarakis SE (1987) Evidence
for reduced recombination on the nondisjoined chromosomes 21 in Down syndrome. Science 237: 652-654 Weening RS, Roos D, Loos JA (1974) Oxygen consumption of phagocytizing cells in human leucocyte and granulocyte preparation: a comparative study. J Lab Clin Med 83 : 570-576 Wisniewsky KE, Wisniewsky HM, Wen GY (1985) Occurrence of neuropathological changes and dementia of Alzheimer's disease and Down's syndrome. Ann Neurol 17: 278-282 Xiao GH, Grzeschik KH, Scherer G (1987) Anonymous DNA sequences from chromosome 21 showing a three allele insertion/deletion RFLP (HGM 9 provisional no D21S82). Nucleic Acids Res 15 : 5499
Received January 18, 1989 / Revised April 10, 1989
