No Significant Effect of Monosomy for Distal 21q22.3 on the Down Syndrome Phenotype in "Mirror" Duplications of Chromosome 21

Constantinos Pangalos1, Didier Théophile2, Pierre-Marie Sinet2, Alexander Marks3, Danai Stamboulieh-Abazis4, Zoubida Chettouh2, Marguerite Prieur1, Christine Verellen5, Marie-Odile Rethoré1, Jérôme Lejeune1, and Jean-Maurice Delabar2

Am. J. Hum. Genet. 51:1240-1250, 1992


Résumé :

Summary : Three Down syndrome patients for whom karyotypic analysis showed a "mirror" reverse tandems duplication of chromosome 21 were studied by phenotypic, cytogenetic, and molecular methods. On high-resolution R-banding analysis performed in two cases, the size of the fusion 21q22.3 band was apparently less than twice the size of the normal 21q22.3, suggesting a partial deletion of distal 21q. The evaluation of eight chromosome 21 single-copy sequences of the 21q22 region - namely, SOD1, D21S15, D21S42, CRYA1, PFKL, CD18, COL6A1, and S100B - by a slot blot method showed in all three cases a partial deletion of 21q22.3 and partial monosomy. The translocation breakpoints were different in each patient, and in two cases the rearranged chromosome was found to be asymmetrical. The molecular definition of the monosomy 21 in each patient was, respectively, COL6A1-S100B, CD18-S100B, and PFKL-S100B. DNA polymorphism analysis indicated in all cases a homozygosity of the duplicated material. The duplicated region was maternal in two patients and paternal in one patient. These data suggest that the reverse tandem chromosomes did not result from a telomeric fusion between chromosomes 21 but from a translocation between sister chromatids. The phenotypes of these patients did not differ significantly from that of individuals with full trisomy 21, except in one case with large ears with an unfolded helix. The fact that monosomy of distal 21q22.3 in these patients resulted in a phenotype very similar to Down syndrome suggests that the duplication of the genes located in this part of chromosome 21 is not necessary for the pathogenesis of the Down syndrome features observed in these patients, including most of the facial and hand features, muscular hypotonia, cardiopathy of the Fallot tetralogy type, and part of the mental retardation.

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Introduction

A rare chromosome variant of trisomy 21 is the "mirror" (reverse tandems) duplication of chromosome 21. Several cases have been described in the literature (Warkany and Soukup 1963; Zellweger et al. 1963; Lejeune et al. 1965; Richards et al. 1965; Cohen and Davidson 1967; Garson et al. 1970; Kadotani et al. 1970; Sachdeva et al. 1971; Vogel 1972; Bartsch-Sandhoff and Schade 1973; Niebuhr 197; Schuh et al. 1974; Hagemeijer and Smit 1977; Harvey 1977; Berg et al. 1980; Cantu et al. 1980; Turleau et al. 1980.

In theory, there are two possible mechanisms for the formation of this chromosomal rearrangement: (1) a telomeric fusion of two chromosomes 21, without loss of chromosomal material, and (2) a reciprocal translocation or exchange between the long arms of chromosome 21 (or sister chromatids). In both cases the deletion or inactivation of one of the two centromeres is necessary for the stability of the derived chro-mosome. Although the precision of the cytogenetic analysis of these chromosomes is limited, in some cases there is apparently little loss of chromosomal material (Turleau et al. 1980), while in others the duplication might be partial, resulting from breakage in the 21q22.3 region, leading to partial monosomy of distal 21q22.3 (Cantu et al.1980). This cytogenetic heterogeneity could account for the clinical variability described in the literature (Niebuhr 1974). The purpose of the present study was to perform the molecular analysis of three cases of trisomy 21 with a mirror duplication of chromosome 21, in order to (1) study the chromosome rearrangement, i.e., determine possible loss of chromosomal material and localize regional breakpoints, and (2) clarify possible genotype-phenotype correlations not only with regard to Down syndrome but also with regard to clinical features observed in cases of partial or possibly complete monosomy 21 (reviewed in Korenberg et al. 1991).

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Subjects and Methods

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Clinical Data

1. Case TY (Greece).

- This girl was born in 1980. She was the first child of healthy parents and was born when her father and mother were, respectively, 26 and 25 years old. Birthweight was 2,250 g (<3d percentile), height was 47 cm (<10th), and head circumference was 31 cm (<3d percentile). At birth the phenotypic evaluation revealed a typical Down syndrome phenotype, although both ears were large with unfolded helix and without crux cymbae (fig. 1). There was no joint hyperflexibility, the tonus was moderately decreased with a diastasis recti, and there was livedo reticularis (marble skin). The findings of the clinical analysis at time of birth are shown in table 1. The child was reexamined at age 9 years (fig. 1 b and table 1). The subject displayed features of typical Down syndrome phenotype, with the exception of ear shape, absence of short neck, and teeth which were normal. No Brushfield spots were present, but the irides were dark. The subject suffered from hyperme-tropia

Psychological and mental evaluation was performed at the age of 10 years 8 mo, by both the WISC-K and Griffith mental development scales. Her overall performance was low. On the WISC-K test, the verbal score was 50, the performance score was 60, and the full-scale score was 50. On the Griffith mental development scale, mental age was 6.4 years, and general quotient was 60.


Fig. 1 - Patient TY at age 1 mo (a) and at age 9 years (b)

2. Case LI (Belgium).

- This boy was born in 1986. He was the first child of healthy parents. At the time of birth of the child, the father was 31 years old, and the mother was 37 years old. Birthweight was 2,660 g (<10th percentile), height was 48 cm (<10th percentile), and head circumference was 32 cm (<3d percentile). At birth the child had features of Down phenotype (fig. 2 and table 1), as well as a murmur accompanied by a discrete cyanosis when she was fed. A tetralogy of Fallot, with subvalvular obstruction, was diagnosed, and a surgical correction was attempted.


Fig. 2 - Patient LI at age 15 d

3. Case AL (France).

- This girl was born in 1963. The case was published by Lejeune et al. (1965). Birthweight was 2,430 g (<3d percentile), height was 46 cm (<3d percentile), and head circumference was 32 cm (<3d percentile). An initial examination at 16 mo revealed a typical Down syndrome phenotype (table 1) with a heart systolic murmur. The auscultation data and the absence of ECG modifications suggested a small ventricular septal defect (Roger disease), which was well tolerated. The IQ (Brunet-Lezine test) was 52. From the age of 6 years, the cardiac auscultation became normal. For this study, the patient was reexamined at the age of 27 years (fig. 3 and table 1). Height was 137 cm, weight was 57 kg, and cranial circumference was 49 cm. The IQ (Binet-Simon test) was 35. The following features were observed: blepharitis conjunctivitis, keratitis, strabisms (operated), severe myopia (-14.5 diopters on both eyes), furrowed tongue, rough voice, and hypothyroidism diagnosed at age 23 years and treated with thyroid hormone. An Epstein-Barr virus-transformed lym-phoblastoid cell line was established.


Fig. 3 - Patient AL at age 27 years

Table I. - Clinical Manifestations of Down Syndrome in Patients TY, LI, and AL
TY LI AL
Birth Age 9 years Age 15 d r Age 16 mo Age 26 years
Jackson's checklist:
Brachycephaly(a)+id ++id
Oblique eye fissure(a)+id++id
Epicanthic eye fold+id++id
Blepharitis, conjunctivitisND+NDND+
Brushfield spots (iris color)- (darks)id-++
Nystagmus-id---
Flat nasal bridge(a)+id+++
Mouth permanently open---ND-
Abnormal teeth -+
Protruding tongue (macroglossia)--+ND-
Furrowed tongue ++
High-arched palate+id-+id
Narrow palates(a)+id-+id
Folded ear (right, left)(a)-,-id-,+-,-id
Short neck(a)+-++-
Loose skin of neck (newborn)ND-ND
Short and broad hands+id++id
Short fifth finger (right, left) +,+id-,--,-id
Incurved fifth finger (right, left)(a)+,+id-,--,-id
Transverse palmar crease (right, left)-,-id-,--,+id
Gap between first and second toes (right, left)(a)+,+id+,++,+id
Congenital heart defect (type)-id+ (Fallot)+ (VSD(b))ND
Murmur-id++-
Joint hyperflexibility-id-+-
Muscular hypotonia(a)+-+++
Total 1212121514
Other features:
Mental retardation (i.e., IQ score) [test]60 [G], 50 [W]51 [BL]35 [BS]
Short stature (height in m) [percentile] -(0.47) [<10th]+(1.2) [<3d]-(0.48) [<10th]+(0.69) [<3d] +(1.37) [<3d]
Head circumference (percentile) 31 cm (<3d)50,5 cm (<50th)32 cm (<3d)ND49 cm (<3d)
Ophthalmological defectHypermetropiaStrabism, myopiaid
Dermatoglyphics:
Palmar t" (right, left)+,+id-,-+,+id
Hypothenar ulnar loop (right, left)+,+id-,-+,+id
High Cummins index > 30 (right, left)-,-id+,-+,-id
Visceral abnormality (other than heart defect)-id--id
Leukemia-----
Alzheimer disease--
NOTE. - id = idem; and ND = not determined. (a) Discriminating signs under age 2 years (Jackson et al. 1976). (b) Ventricular septal defect. (c) G = Griffith mental development scale; W = WISC-K test; BL. = Brunet-Lezine test; and BS - Binet-Simon test.

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Cytogenetic Analysis

Seventy-two-hour lymphocyte culture was performed in all cases. Standard banding techniques - i.e., RHG and GTG, C and NOR, and RTBG (high resolution R-banding) - were performed. In all three cases the karyotypic analysis showed a mirror duplication of chromosome 21:46,XX, or XY, -21, + psu dic(21)t(21;21)(q22.3;q22.3). Two centromeres at both ends of the rearranged chromosomes were revealed, by the C-banding technique, with apparent inactivation of one of the two centromeres. The duplicated chromosome had stalks in both arms and satellites visualized by the NOR technique (fig. 4).

Lymphocytes from cases TY and AL were subjected to high-resolution R-banding analysis. In AL, the size of the fusion 21q22.3 band was less than twice that of the 21 q22.3 band, suggesting a partial deletion of 21q22.3. In all cases, the karyotype of the parents was apparently normal, as assessed by standard banding techniques.

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Molecular Analysis

The copy numbers of chromosome 21 single-copy sequences were evaluated by a slot blot hybridization method described elsewhere (Rahmani et al. 1989; Blouin et al. 1990). DNA was purified from blood, by standard techniques. Varying amounts of denaturated DNA from a normal control (C), a trisomy 21 patient (D), and the subject to be analyzed (X) were loaded onto a nylon membrane. Successive hybridizations were carried out with both a reference probe and chromosome 21 probe (32PdCTP-labeled inserts. Intensities of the signals on autoradiograms were quantified by densitometric scanning (Shimadzu TLC CS 930 scanner). Linear regressions between reference and chromosome 21 probe signals for C, D, and X were computed, and the conclusion that the DNA from the studied subject X has one, two, or three copies for a given chromosome 21 sequence was assessed by statistical comparison of its regression slope with those of C and D. Figure 5 summarizes the results of duplicated estimations of the copy number of eight sequences of the 21q22.1-q22.3 region -namely, SOD1 (superoxide dismutase 1) (Lieman-Hurwitz et al, 1982), D21S15 (Stewart et al., 1985), D21S42 (Korenberg et al. 1987), CRYA1 (Crystallin, alpha polypeptide 1) (McDevitt et al. 1986), PFKL (phosphofructokinase, liver typed) (Levanon et al. 1987), CD18 (antigen CD18, lymphocyte function-associated antigen 1) (Kishimoto et al. 1987), COL6A1 (collagen, type VI, alpha 1) (Weil et a1.1988), and S100B (S100 protein, beta polypeptide, neural) (Allore et al, 1988). The normal order and localization of these sequences on chromosome 21, from centromere to telomere, are SOD1 on 21q22.1, D21S15, D21S42, CRYA1, PFKL, CD 18, COL6A1, and S100B on 21q22.3 (Burmeister et al. 1991; Cox and Shimizu 1991; Crété et al. 1991; Petersen et al. 1991; Delabar et al. 1992; Tanzi et al. 1992; Wang et al. 1992). Table 2 gives typical data obtained for copy-number determinations of three sequences from each patient.

In patient TY, the copy numbers for SOD1, D21S15, D21S42, CRYA1, PFKL, CD18, COL6A1, and S100B were 3, 3, 3, 2, 1, 1, 1, and 1, respectively, thus indicating that the translocation breakpoint lies within the 21q22.3 region, between markers D21S42 and PFKL, but at different loci on each of the two chromosomes involved in the duplication: between D21S42 and CRYA1 on one chromosome 21 and between CRYA1 and PFKL on the other. In patient LI, the copy numbers for the same eight probes were 3, 3, 3, 3, 2, 1, 1, and 1, respectively, thus indicating that the translocation breakpoint lies within the 21q22.3 region, between markers CRYA1 and CD 18, but again at different loci on each of the two chromosomes 21 involved in the duplication: between CRYA1 and PFKL on one chromosome 21 and between PFKL and CD18 on the other. In patient AL, the copy numbers for the eight probes were 3, 3, 3, 3, 3, 3, 1, and 1, respectively. The breakpoint of the translocation is thus more distal than it is in patients TY and LI and is located between markers CD 18 and COL6A1.

DNA polymorphism analysis was performed, using blood DNA of the patients and their parents, by PCR amplification. The PCR polymorphisms were a (GT)n repeat of locus 21-GT14 (D21S215) close to the centromere on 21q (Williamson et al. 1991; S. E. Antonarakis and A. C. Warren, personal communication), a (ATTT)n repeat of the Alu VpA at IVS5 of the IFNAR gene (McInnis et al. 1991), a (GT)n repeat of locus D21S167 (Guo et al. 1990), and a (GT)n repeat of locus PFKL (Polymeropoulos et al. 1991). One of the pair of primers was labeled by adjunction of fluorescein. PAGE was performed using an ALF DNA sequencer (Pharmacia). The copy number of alleles for the loci studied was scored by comparing the patterns and intensities of allele peaks in the patients and their parents (J.-M. Delabar and D. Théophile, unpublished data). Results in table 3 indicate that parental origin of the duplicated chromosome 21 was paternal in patient TY and maternal in patients LI and AL. In the three patients the data were consistent with a homozygosity of the duplicated alleles. Furthermore, in patient TY the deletion of the paternal alleles confirmed the monosomy for PFKL.

Table 2 - Quantification of Chromosome 21 Sequences in Patients TY, LI, and AL
Patient and Chromosome 21 Probe(y) / Reference Probe(x) Values of Slopes F ± SD of Regression Lines (a) (y = Fx) 95 % Confidence Interval of Difference between Slopes (a,b) Conclusion
TY:
D21S42/COL1A2C1 = .84 ± .07D1-C1 = + .50 to +1.76D > C; difference 3:2
C2 = .38 ± .03D2-C2 = + .09 to + .34
D1 = 1.96 ± .23X1-C1 = + .57 to + 1.26X > C
D2 = .60 ± .05X2-C2 = + .13 to + .45
X1 = 1.75 ± .11D1-X1 = - .52 to + .94D = X; three copies
X2 = .68 ± .06D2-X2 = - .24 to + .08
CRYA1 /COL1A2C1 = 1.75 ± .21D1-C1 = + .67 to + 2.07D > C; difference 3:2
C2 = 1.70 ± .08D2-C2 = + 1.67 to + 2.41
D1 = 3.12 ± .17X1-C1 = - .33 to + .79X = C; two copies
D2 = 3.74 ± .17X2-C2 = - .04 to + .63
X1 = 1.98 ± .17D1-X1 = + .46 to + 1.83D > X
X2 = 2.00 ± .08D2-X2 = + 1.37 to + 2.12
PFKL/COL1A2C1 = 1.28 ± .09D1-C1 = + .61 to + 1.36D > C; difference 3:2
C2 = 1.64 ± .09D2-C2 = + 1.06 to + 2.21
D1 = 2.23 ± .16X1-C1 = - .94 to - .53X < C; one copy
D2 = 3.28 ± .29X2-C2 = - 1.34 to - .75
X1 = .51 ± .OSD1-X1 = + 1.42 to + 2.03D > X
X2 = .60 ± .08D2-X2 = + 2.07 to + 3.30
LI:
CRYA1/COL1A1 C1 = .86 ± .11D1-C1 = +.51 to + 1.43D > C; difference 3:2
C2 = .65 ± .09D2-C2 = + .38 to + 1.17
D1 = -1.83 ± .20X1-C1 = + .29 to + 1.34X > C
D2 = 1.43 ± .19X2-C2 = + .39 to + 1.26
X1 = 1.67 ± .24D1-X1 = - .51 to + .82D = X; three copies
X2 = 1.48 ± .21D2-X2 = - .64 to + .54
PFKL/COL1A1 C1 = 3.91 ± .36D1-C1 = + 1.11 to + 3.60D > C; difference 3:2
C2 = 1.01 ± .12D2-C2 = + 0.20 to + 1.00
D1 = 6.27 ± .46X1-C1 = - 1.93 to + .19X = C; two copies
D2 = 1.62 ± .14X2-C2 = - .57 to +.10
X1 = 3.05 ± .33D1-X1 = + 2.06 to + 4.38D > X
X2 = .77 ± .08D2-X2 = + .52 to + 1.16
CD18/COL1A1 C1 = 3.56 ± .10D1-C1 = + 2.53 to + 3.61D > C; difference 3:2
C2 = 3.16 ± .30D2-C2 = + 3.06 to + 4.69
D1 = 6.63 ± .26X1-C1 = - 2.55 to - 1.99X < C; one copy
D2 = 7.04 ± .23X2-C2 = - 2.84 to - 1.28
X1 = 1.28 ± .06D1-X1 = + 4.81 to + 5.87D > X
X2 = 1.10 ± .07D2-X2 = + 5.40 to + 6.47
AL:
PFKL/C0L1A1 C1 = .70 ± .03D1-C1 = + .22 to + .59D > C; difference 3:2
C2 = 1.53 ± .12D2-C2 = + .40 to + .79
DI = 1.11 ± .08X1-C1 = + .12 to + .40X > C
D2 = 3.58 ± .28X2-C2 = + .19 to + .56
X1 = .96 ± .O5D1-X1 = - .O5 to + .35D = X; three copies
X2 = 4.45 ± .06D2-X2 = - .07 to + .48
CD18/C0L1A1C1 = .59 ± .O5D1-C1 = + .54 to + .85D > C; difference 3:2
C2 = .52 ± .03D2-C2 = + .32 to + .83
D1 = 1.28 ± .O5X1-C1 = + .32 to + .70X > C
D2 = 1.10 ± .12X2-C2 = + .26 to + .57
X1 = 1.10 ± .07D1-X1 = - .02 to + .39D = X; three copies
X2 = .93 ± .O5D2-X2 = - .08 to + .42
C0L6A/C0L1A2 C1 = .53 ± .06D1-C1 = + .26 to + .59D > C; difference 3:2
C2 = .36 ± .02D2-C2 = + .54 to + 1.16
D1 = .96 ± .04D1-C1 = - .53 to - .31X < C; one copy
D2 = 1.21 ± .16D2-C2 = - .28 to -.20
X1 = .11 ± .O1D1-X1 = + .76 to + .92D > X
X2 = .12 ± .01D2-X2 = + .79 to + 1.39
(a) Numbers after C, D, and X refer to independent experiments different membranes). (b) For two compared slopes, when the 95 % confidence interval of their difference contains the zero value the slopes are not statistically different, whereas when the confidence interval does not contain the zero value the slopes are different (for more details on the method, see Blouin et al. 1990).
Table 3 - DNA Polymorphism Analysis in Families of Patients TY, Ll, and AL
Genotype (b) of
Family of TY Family of LI Family of AL
Locus, LOCALIZATION (a) Father Mother Child Father Mother Child Father Mother Child
D21S215(GT14), 21q11131213312231331212112
IFNAR, 21q22.2121312212131121233233
D21S167, 21q22.2231212234122242312112
PFKL, 21q22.323141NDNDND2312223
(a) The regional localization of the loci is according to the consensus maps of Cox and Shimizu (1991) and Williamson et al. (1991). (b) The numbers represent the different alleles at a specific locus.


Figure 4. - Top, Karyotypes showing the banding patterns of patients AL, TY, and LI, by using the standard techniques RHG, GTG, CGB, and NOR. Left, High-resolution R-banding (RTBG) analysis of patients AL and TY.


Figure 5. - Results of the dosage analysis of tested sequences (no. of copies) in patients TY, LI, and AL.

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Discussion

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Chromosome Rearrangement

Cytogenetic and molecular analyses of partial duplications or deletions of chromosome 21 are useful for studies on phenotype-genotype correlations in Down syndrome (Epstein 1990. Such cases may include patients with tandem or reverse tandem translocations of the long arm of chromosome 21. Most cases described in the literature (Niebuhr 1974; Cantu et al. 1980; Turleau et al. 1980) are reverse tandem (i.e., mirror) translocations of all or part of the long arm of the chromosome. We performed combined clinical, cytogenetic, and molecular analysis of three patients with Down syndrome resulting from such a mirror duplication of chromosome 21. There are two theoretical mechanisms for such chromosomal rearrangements: first, a telomeric fusion of two chromosomes 21 after opening of telomeric palindromes, without loss of chromosomal material, and, second, a translocation between the long arms of two homologous chromosomes 21 or between the two chromatids of the same chromosome 21. If the translocation breakpoints are at the same sites in each chromosome or chromatid - i.e., are "homotopic" breaks - the resulting chromosome would be symmetrical. If the translocation breakpoints are at different sites - i.e., are "heterotopic" breaks - the derived chromosome would be asymmetrical. The derived chromosome is stable only if one of the two centromeres is deleted or inactivated. In two of the cases, TY and AL, high-resolution banding was performed. The fused 21q22.3 band was less than twice the size of the 21q22.3 band, suggesting a partial deletion. In all cases, one of the two centromeres was inactivated.

The copy numbers of eight chromosome 21-specific sequences indicate partial deletion of the region 21q22.3, in all cases. The extent of the deletion is different in each case: the deletion in TY is largest, that in LI is intermediate, and that in AL is smallest. Moreover, the translocation breakpoints on the two chromosomes 21 involved in the minor duplication are located in homologous regions in case AL and in different regions in cases TY and LI. Thus, AL has undergone a homotopic translocation giving a symmetrical derived chromosome leading to partial trisomy 21 and partial monosomy of 21q22.3 without disomy. Conversely, in cases TY and LI, the translocation is heterotopic, and the derived chromosomes are asymmetrical, leading to partial trisomy 21, partial monosomy of 21q22.3, and disomy for the CRYA1 and PFKL regions, respectively.

DNA polymorphism analysis shows that the parental origin of the duplicated chromosome material is paternal in patient TY and maternal in patients LI and AL. The loss of the two paternal PFKL alleles in patient TY strongly suggests that the mirror chromosome is formed by two elements of paternal origin. In all cases, data were consistent with the duplicated alleles being homozygous. Therefore, the simplest mechanism leading to the mirror chromosome would be a translocation for fusion) between sister chromatids, occurring at a prezygotic stage. This type of rearrangement at a postzygotic stage is less likely, since no normal and/or ( - 21 ) cells were observed in the karyotype of the patients. There is a possible alternative mechanism: translocation between two chromosomes 21 in a trisomy 21 zygote at a very early stage of the postzygotic development.

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Phenotype-Genotype Correlations with Regard to Partial Trisomy 21

The phenotype of all three patients was not significantly different from that of patients with trisomy for the whole chromosome 21. Table 1 gives, for each patient, the total number of signs on Jackson's checklist that were observed. At an early age, TY, LI, and AL had 12, 12, and 15 signs, respectively, which correspond to 84 %, 84 %, and 100 % probability of trisomy 21. When examined later, TY and AL had 12 and 14 signs, respectively, which correspond to 84 % and 100 % probability of trisomy 21. The 10 most discriminating signs of Down syndrome for trisomy 21 patients younger than age 2 years (table 1) were scored for each patient, and their respective discrimination coefficients were summed; the results were .1137 for TY, .0956 for LI, and .1119 for AL. These values are typical scores for trisomy 21 and do not fall within the overlap area between normal and Down syndrome subjects (Jackson et al. 1976). Transverse palmar creases were not present in TY and LI. Muscular hypotonia was moderate in TY and AL. These characteristics are within the limits of the variability of the phenotypic expression of Down syndrome, and in no case could the absence of a Down syndrome feature in a patient with partial trisomy 21 be interpreted as resulting from nonduplication of a particular region, since none of these features except mental retardation is expressed in 100 % of patients with full trisomy 21. The presence of Down syndrome features in these patients strongly suggests that the genes involved in their pathogenesis are localized proximally to the translocation breakpoints in 21q22.3. Thus, the trisomy 21 signs present in TY and/or LI (table 1) presumably result from the duplication of genes proximal to CRYA1 and/or PFKL. These signs include many of the facial and hand features, the gap between the first and second toes, the congenital hear disease with tetralogy of the Fallot type, muscular hypotonia, mental retardation, and short stature. Similarly, other signs not present in TY and LI but present in AL, such as Brushfield spots, abnormal teeth, joint hyperlaxity, and ventricular septal defect (table 17, are probably linked to the duplication of genes proximal to COL6A1. Some of the Down syndrome features observed in these three patients, including flat nasal bridge, macroglossia, incurved fifth finger, hypotonia, short stature, and mental retardation, have been associated with the duplication of the region around D21S55 ("Down syndrome chromosome region") (Rahmani et al. 1989,1990; Sinet et al. 1991), located between D21S17 and ETS2, on 21q22.2-q22.3 proximal. The central role of the duplication of this region in the pathogenesis of Down syndrome has also been reported by others (Korenberg et al. 1989, 1992). This region, proximal to D21S15, does not overlap the PFKL-telomere region. Other facial and hand features, as well as mental retardation, have been observed in Down syndrome patients with molecularly defined duplications of 21q22.3 (McCormick et al. 1989; Korenberg et al. 1990b, 1992; Petersen et al. 1990). Our results strongly suggest that duplication of the genes of the PFKL-21qter region is not necessary for the generation of these features.

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Phenotype-Genotype Correlations with Regard to Partial Monosomy 21

Finally, these patients did not have the following phenotypic features typical of monosomy 21: hypertonia, high nasal bridge, down-slanted palpebral fissure, and prominent occiput. Such features have been described in karyotypically full or partial monosomies 21, including ring chromosome 21 and, in some cases, have been defined at the molecular level (Pellissier et al. 1987; Phelan et al. 1988; Chettouh et al. 1991; Korenberg et al. 1991). It is interesting that individuals with ring chromosome 21 have been reported without phenotypic anomaly (reviewed in Dallapiccola et al. 1986). These cases have a monosomy for the distal part of 21q22.3 (Korenberg et al. 1990a, McGinniss et al. 1992). Further molecular characterization of normal individuals with ring chromosome 21 should define the maximum monosomy which does not result in phenotypic modifications. Of the three patients described above, only TY had one sign which could be interpreted as a phenotypic expression of monosomy 21 - i.e., large ears with unfolded helices. Except for this specific feature, monosomy of distal 21q22.3, from the telomere to PFKL, had no significant effect on the expression of Down syndrome phenotype. This adds further weight to the suggestion that the genes contained in this region - approximately the distal third of 21q22.3, i.e., 1.5-3 Mb (Burmeister et al. 1991; Wang et al. 1992) - are not necessary for the expression of the Down syndrome features, including most of the facial and hand features, short stature, muscular hypotonia, cardiopathy of the Fallot-type tetralogy, and part of the mental retardation, which are found in our patients.


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