Down syndrome, the most frequent birth defect (around 1/700 births), results in most cases from the presence in all cells of an extra copy of chromosome 21. It is characterized by a complex and specific phenotype with mental retardation. One step towards the identification of genes that could play a role in the pathogenesis of the disease is the definition, as concisely as possible, of minimal regions on chromosome 21 involved in producing particular features of the phenotype [1]. This can be done by analysing genotype-phenotype correlations in rare patients with partial trisomy 21. Thus, karyotypic studies have indicated that trisomy of the distal part alone of chromosome 21, namely band q22, is responsible for Down syndrome [24]. More recently, molecular analysis of some patients [57] has confirmed this proposition and we have suggested that the region around probe D21S55 is critical in the phenotypic expression of trisomy 21 [8].

We report the analysis of the genotype-phenotype correlations in 10 patients with partial trisomy 21 for different segments of chromosome 21 leading to the molecular mapping of 24 Down syndrome features on chromosome 21. Interestingly, 13 (more than half) of these features, including a major contribution to mental retardation, map to the single region D21S55 (Down syndrome chromosome region; DCR) located on sub-band 21q22.2 or very proximal 21q22.3. Six other features map to the region D21S55-MX1, including the DCR and spanning sub-band 21q22.2 down to the proximal quarter of 21q22.3. Thus, the clustering of most of the Down syndrome features within a relatively small region of chromosome 21 suggests that only one or a few genes belonging to this region are required to produce this complex phenotype.

Material and Methods

Karyotypic Analysis

High-resolution R and G banding were performed on 72-hour lymphocyte cultures from all patients and previously reported karyotypic analyses in patients ML and DL [3, 9] were reassessed (table 1).

Table 1 Patients with partial trisomy 21.

Phenotypic Analysis

The phenotypic analysis of the patients was based on the assessment of a total of 33 features commonly encountered in Down syndrome patients: 25 features on the phenotypic checklist of Jackson et al. [10] and 8 other features including IQ, short stature and dermatoglyphic characteristics [11]. For patients FG, IG, TY, LI and AL the phenotypic analysis has been previously reported [11, 12]. For patiens ML [9], DL [9] and AP [13], the checklist was scored from previously published phenotypic descriptions whereas for other patients direct clinical examination was performed.

Molecular Analysis

The copy numbers of chromosome 21 single-copy sequences were evaluated by a slot blot hybridization method as previously described [8, 14]. Briefly, varying amounts of denatured blood DNA from a normal control (C), a trisomy 21 subject (D) and the patient to be analysed (X) were loaded onto a nylon membrane. The membrane was successively hybridized with a reference probe and chromosome 21 probes (32PdCTP-labelled inserts). Intensities of the signals on autora-diograms were quantified by densitometric scanning (Shimadzu TLC CS 930 scanner) and linear regressions between reference and chromosome 21 probe signals for C, D and X were computed. The conclusion that the DNA from the studied patient had 2 or 3 copies for a given sequence was assessed by statistical comparison of its regression slope with those of C and D. All gene dosage evaluations were done in duplicate. The following 25 probes were used: APP, BCEI, CBS, CD 18, COL6A1, CRYA1, ETS2, MX1, PFKL, S100B and SOD1 cDNAs and the anonymous DNA probes D21S1, D21S11, D21S13, D21S15, D21S16, D21S17, D21S19, D21S42, D21S52, D21S54, D21S55, D21S58, D21S59 and D21S65 [15]. For some patients, full (patient FG [16]) or partial (patients IG [8, 17], AB, ML and TY, LI, AL [12, 17]) molecular analyses have been previously reported.


Molecular Definition of Partial Trisomies 21

The 10 patients studied each bear a partial trisomy 21 which was first evidenced by cytogenetic analysis. Table 1 gives the results of the high-resolution chromosome examinations. The origins of these partial trisomies 21 were diverse: de novo duplication of chromosome 21 (patients FG and IG), de novo translocation (21;21) (patient AB), trisomy for a chromosome 21 with a de novo interstitial deletion (patient SC), trisomy resulting from the malsegregation of a balanced parental translocation (sibs ML and DL, and patient AP), de novo mirror duplication of chromosome 21 (patients TY, LI, and AL).

We defined the extent of these partial trisomies 21 at the molecular level by estimating the copy number of 25 chromosome-21 -specific and unique sequences in blood DNA of the patients, using a slot blot method previously described [14] (fig. 1). For patient DL [9], molecular analysis was not possible. However, as DL and his sister ML inherited complementary extra chromosome 21 segments resulting from the malsegregations of the maternal (15;21) balanced translocation, the extent of the DL duplication was assumed to be the complementary part of the ML duplication defined by gene-dosage. Similarly, patient AP was not accessible to gene dosage analysis, but the extent of his duplication could be deduced from the analysis of his sister [17] who had a monosomy for the complementary part of chromosome 21 resulting from the malsegreTYon of the maternal (X;21) balanced translocation [13].

Fig. 1
figure 1

Molecular definitions of the partial trisomies 21. Boxes represent the regions of chromosome 21 present in triplicate in the patients: FG = D21S16-D21S55; IG = D21S55-MX1; AB = D21S55-qter; SC = pter-D21S54 and D21S55-qter; ML = pter-D21S54; DL = SOD1-qter; AP = D21S52-qter; TY = pter-D21S42; LI = pter-CRYA1; AL = pter-CD18. The order and regional localization of probes on chromosome 21 are consistent with recent genetic and physical mapping data [1724].

Given the regional localization and order of the probes established by recent genetic [18, 19] and physical [17, 2024] mapping of chromosome 21, the results of the molecular analysis were consistent with cytogenetic observations. Except for the mirror duplications in which terminal delections, i.e., monosomies of distal 21q, were observed [12], other trisomies were apparently devoid of complex rearrangements.

Phenotypic Analysis

Table 2 shows the results of the phenotypic analysis of the patients based on the assessment of a total of 33 features commonly encountered in Down syndrome. None of them had visceral abnormalities other than heart defect, nor a previous history of leukaemia. Moreover, the patients were too young to present clinical symptoms of dementia of the Alzheimer type. Three patients had a congenital heart defect. A severe and unidentified cardiopathy in patient DL caused his death shortly after birth [9]. Patient LI had a Fallot tetralogy and patient AL suffered from a ventricular septal defect.

Table 2 Phenotypic analysis of the patients

The Jackson phenotypic scores for the clinical diagnosis of trisomy 21 [10] indicated a 100% probability of trisomy 21 in patients FG and AL, whereas the scores were within the area of overlap between normal and trisomy 21 subjects for patients IG, AB, SC, DL, TY and LI, with 5–12 signs. Two patients, ML and AP, had a score in the normal range. This result for patient ML is consistent with the established opinion that trisomy for the proximal half of chromosome 21 does not induce the Down syndrome phenotype. In patient AP, the normal Jackson score probably results from X inactivation spreading to the X-translocated chromosome 21 fragment, as evidenced by previously reported replication studies on lymphocytes [13]. However, this patient had some of the dermatoglyphic features of Down syndrome.

All patients, except patient AB, had a mental delay as assessed by global IQ tests. In a recent survey of non-institutionalized trisomy 21 patients [M. Prieur et al., unpubl. data], the mean (± SD) IQ in 21 patients between 1 and 3 years old was found to be 76 ± 20, with 5 patients scoring100. Thus, possibly due to improvements in the educational training of the children by their families, a normal IQ at this age is not exceptional in full trisomy 21. Therefore, the mental-development status of patient AB cannot be accurately predicted from this early IQ evaluation and deserves follow up after early infancy. Patients ML and AP had moderate mental retardation, their IQ being 2 DS above the mean IQ of trisomy 21 patients at the same age [25].

Genotype-Phenotype Correlations

The genotype-phenotype correlations were analysed including only features that were present (positive sign) in at least 2 of the patients. Thus, blepharitis, conjunctivities, nystagmus, mouth permanently open, abnormal teeth, loose skin of neck, visceral abnormality other than heart defect, leukaemia and Alzheimer’s disease were not included in this analysis. Moreover, heart murmur associated in 3 patients with a congenital heart defect was not considered to be an individual feature distinct from heart defect. Thus, 24 features were taken into consideration and their presence correlated to the genotypic modifications observed in the patients. For each of these specific features it was possible to define a minimal region [1] of chromosome 21 which was present in 3 copies in all patients expressing this feature. For example, flat nasal bridge was observed in 7 patients, namely FG, IG, AB, DL, TY, LI and AL; the minimal region present in triplicate in every one of these patients is the region containing probe D21S55 and surrounded by probes D21S17 and ETS2. Twenty-four features of Down syndrome were thereby assigned to 6 minimal regions (fig. 2). Their positions on chromosome 21 were determined from recent physical maps [17, 2024]. A seventh region was added and corresponds to the moderate mental retardation observed in patient ML. Two regions were found to be particularly interesting: D21S55, the minimal region for the phenotypic expression of 13 features (short stature, muscular hypotonia, joint hyperflexibility, 9 morphological signs and contribution to mental retardation), and D21S55-MX1, the minimal region for the expression of 6 morphological features.

Fig. 2
figure 2

Molecular mapping of 24 features of Down syndrome on chromosome 21. For each feature the minimal region of chromosome 21 (region 1–6) was defined as the region which was triplicated in every one of the patients expressing that feature. Region pter-D21S54 corresponds to the chromosome 21 segment trisomic in patient ML. The order and regional localization of probes on chromosome 21 are consistent with recent genetic and physical mapping data [1724].


We have mapped the Down syndrome phenotype on chromosome 21 (fig. 2) and thereby confirmed that this phenotype mostly results from the trisomy of the 21q22 band. More precisely, our data indicate that trisomy of the segment of chromosome 21 from SOD1 to CRYA1 contributes to the expression of 22 of the 24 mapped features. Inversely, the proximal part of the chromosome from pter to D21S54 and the distal part from PFKL to S100B play no major role in the pathogenesis of the Down syndrome phenotype.

Molecular definitions of chromosome 21 regions contributing to the Down syndrome phenotype have been previously reported, using data from single or a small number of patients with partial trisomy 21. The definition of the D21S55 region [8, 11], involved in the expression of short stature, components of the facial, hand and foot dysmorphies, muscular hypotonia and contributing to the mental retardation, led to the proposal that this limited region of chromosome 21, named the Down syndrome chromosome region [11, 26] or DCR [15], plays a crucial role in the pathogenesis of Down syndrome. This is consistent with data indicating that the region D21S13-D21S58 [5] including APP and SOD1 [6] does not significantly contribute to most Down syndrome features, whereas trisomy of the region between D21S58 and D21S55 down to COL6A1 [5] or of the region distal to D21S17 [27] was responsible for the characteristic facial changes, congenital heart disease and mental retardation. A recent study [7] assigned some of the facial features and incurved 5th finger to the region D21S55-D21S15, the brachycephaly, the gap between 1 st and 2nd toes, joint laxity, hypotonia, gut atresia and mental retardation to the region D21S8 (located between D21S11 and APP)-D21S15, and the dermatoglyphics, congenital heart disease of the endocardial-cushion-de-fect type, and mental retardation to the D21S5 5-qter region.

Variability in phenotypic expression is one of the characteristics of trisomy 21. It is therefore expected that patients with partial trisomy 21 including gene(s) responsible for a specific feature may not express this feature. To overcome this problem and increase the power of the genotype-phenotype correlation analysis, data from 10 patients with overlapping partial trisomies 21 were pooled. Our data are consistent with previous data and significantly increase the number of features of the Down syndrome phenotype mapped on chromosome 21 (fig. 2). The D21S55 region, or DCR, includes determinants for 9 morphological signs and 4 major features (short stature, joint hyperlaxity, muscular hypotonia and participation in mental retardation). Physical-mapping data and fluorescent in situ hybridization experiments with a D21S55 cosmid on prometaphasic chromosomes [D. Theophile, unpubl. data] indicate that this region is localized on 21q22.2 and proximal 21q22.3. Its size has been estimated to be between 400 kb and 3 Mb [8]. There is only one known gene, ERG [28], physically linked to D21S55 [21], that might belong to the DCR. However, this gene is not duplicated in patient FG [29], indicating that the distal boundary of the DCR is proximal to it. The D21S55-MX1 region (region 2), which includes the DCR, is the minimal region for 6 facial and dermatoglyphic features. From the regional mapping of the probes defining this region and the physical cartography of distal 21q [22], its maximum size can be estimated to be 6 Mb spanning 21q22.2 down to the proximal quarter of 21q22.3. The genes ERG [28], ETS2 [30], HMG14 [31] and MX1 [32] map in this region [33] and are therefore potential candidates for roles in the pathogenesis of these features. As DCR is part of region 2, our results indicate that genes contained in this relatively small part of chromosome 21 from D21S55 to MX1, when triplicated, contribute to most of the phenotypic expression of Down syndrome (19 of the 24 studied features). Furthermore the DCR and region 2 overlap, so some of the features assigned to region 2 might in fact map in the DCR. Similarly, short 5th finger maps in region 3: SOD1-D21S55, short neck in region 4: SOD1-CRYA1, furrowed tongue in region 5: D21S16-D21S55 and brachycephaly in region 6: pter-D21S42, all regions overlapping the DCR and may be in fact mapping within it. Finally, the minimal region for brachycephaly, which we define as pter-D21S42, can be reduced to D21S8-D21S55[7].

Several types of congenital cardiopathies are observed in Down syndrome, afflicting around 40% of the patients: atrioventricular canal, ventricular septal defect, patent ductus arteriosus, atrial septal defect, Fallot tetralogy and other anomalies in decreasing order of frequency. Mapping each of these individual defects on chromosome 21 might help answer the question whether a different gene (or set of genes) is involved in the generation of each type of defect or if there are pathogenic mechanisms common to several, if not all, of these defects. As the phenotypic similarities between the heart defect in patient DL and that in patients LI and AL could not be assessed, it was not possible to define a minimal region for a specific type of congenital heart disease from our data. However, as patients LI and AL are not trisomic for the regions PFKL-S100B and COL6A1-S100B, respectively, it is likely that these regions do not play a significant role in the pathogenesis of Fallot tetralogy and ventricular septal defect. If we assume that the overdosage of one gene or of a set of contiguous genes may be responsible for the various types of heart anomalies, then we can define the region SOD1-CRYA1 (region 4) as the minimal region for congenital heart disease. With this assumption and the previously reported mapping of the heart defect of the endocardial cushion type to the region D21S55-qter [7], the minimal region for congenital heart disease can be reduced to D21S55-CRYA1.

Our data suggest that more than one region is involved in the pathogenesis of mental retardation in Down sydrome: all patients presented a mental delay except subject AB, who was too young for evaluation of mental status. AP and ML were moderately retarded as compared to trisomy 21 individuals of the same age [25]. As previously discussed, the weak Down syndrome phenotype in patient AP probably results from a spreading of the X inactivation to the X-translocated chromosome 21, which may include genes involved in mental retardation. In patient ML, although one cannot exclude that the trisomy for the distal sub-band 15q26.2 could contribute to the mental delay, the overdosage of gene(s) in the region pter-D21S54 may also be involved. Other observations suggest that the contribution of the proximal part of chromosome 21 to mental retardation might be relatively minor: two patients, one trisomic for the region D21S13-D21S8 (located between D21S11 and APP) [34] and the other trisomic for the region D21S13-D21S58 [35] were reported to have a subnormal mental development and a very slight mental delay, respectively. These data are consistent with cytogenetic studies concluding that trisomy for the proximal part of chromosome 21 including band 21q21 had either no significant [36, 37] or moderate [38, 39] effects on mental development. Patient IG had a mental retardation with a global cognitive defect, language impairment and behaviour expected in Down syndrome [4]. Thus, the trisomy of the region D21S55-MX1 is apparently determinant for the pathogenesis of mental retardation. Moreover, as patient FG also had a mental impairment characteristic of Down syndrome [40], it may be that other genes are involved in mental retardation in addition to the moderate contribution of the proximal 21q. These genes might be duplicated in both patients FG and IG, and therefore located in the DCR.

In conclusion, the molecular mapping of the Down syndrome phenotype shows that most of the features are clustered on two relatively small regions of chromosome 21, D21S55 (DCR) and D21S55-MX1 on sub-bands 21q22.2 and 21q22.3. No gene has yet been identified in the DCR and four genes belong to the D21S55-MX1 region, namely ETS2, ERG, HMG14 and MX1 which thus become candidates for involvement in the phenotypic expression of trisomy 21. Other genes will undoubtedly be identified in these two regions and it is probable that one or some of them, when triplicated, play a major role in the pathogenesis of the complex phenotype that constitutes Down syndrome.