We present 41 cases in this study, including 30 with partial aneuploidy for chromosome 21 (): 19 cases of partial trisomy 21 (12 new and seven previously reported cases)7, 8, 18, 37, 38, 39
and 11 cases of partial monosomy 21 (eight new and three previously reported cases).24, 40, 41, 42
We also studied five cases presenting a DS-like phenotype with a normal karyotype. The remaining six trisomy 21 cases consisted of two t(21;21) and one duplication involving HSA21 tested to identify possible partial trisomy and three free trisomy 21 cases used as controls. The clinical features of the 41 cases were collected from the original medical records given by the referring physician. The available data and clinical evaluations are summarized in and .
All 41 cases were analyzed by aCGH on the HSA21q BAC array ( and ). The results by aCGH were concordant with cytogenetic observation in all cases. Validation by real-time quantitative PCR was carried out for 20/30 (64%) of cases with partial aneuploidy, and confirmed the aCGH results ().
Figure 1 Examples of HSA21 array CGH results. (a) Plots of DNA ratio for two cases. X-axis, position along HSA21; Y-axis, normalized log2 ratio of case/control signal. (b) Data from a segment using CGH-explorer.35 The examples show cases of partial trisomy (left (more ...)
aCGH results. X-axis represents position along the HSA21q in Mb; Y-axis represents the cases. Shading represents ploidy along the chromosome as defined in the key. Two array clone gaps are visible in each sample.
We investigated four cases (27–30), which presented with the aspects of a DS phenotype, two of which had karyotypes with abnormalities involving HSA21. Case 27 was investigated because of the presence of several features of DS, and a tetrasomy 18p karyotype, which might have masked a cryptic trisomy 21. However, aCGH revealed a normal HSA21 content. Cases 28–30 had some features of DS leading us to suspect a possible chromosome 21 cryptic duplication. Again, aCGH analyses revealed a normal HSA21 content for all three cases. Case 26 was included as normal control with a balanced t(21;21) and normal phenotype.
Despite the phenotypic variability between cases of full and partial monosomy 21 reported in the literature, features are frequently described include intrauterine prenatal and postnatal growth retardation, down-slanted palpebral fissures, low set ears, hypertonia, heart defect and mental retardation.6
We report the clinical findings of 7/12 cases for partial monosomy 21. All the cases reported (7/7) were described with mental retardation. Short stature is present in 4/5 cases of partial monosomy 21. Large ears, large nose, broad mouth and hypertelorism are frequently associated with partial monosomy 21, each with 3/4 cases presenting the phenotype when assessed.
Case 39 was reported with subtle dysmorphic anomalies including marfanoid habitus. The thin marfanoid build was previously associated with distal monosomy 21q.4, 24
The breakpoints of the different partial aneuploidies appear to be nonrandomly distributed along the length of 21p. If one divides 21q in the bins of 5
Mb, the segments between 30–35
Mbs and 35–40
Mbs contain 10 and 16 breakpoints (), respectively; in contrast, all other such segments contain 0–6 breakpoints each. This distribution of breakpoints result in a P
value=0.0064 (Fisher's exact test). This could be the result of an ascertainment bias because the cases have been collected based on phenotypic consequences; alternatively, this could reflect a differential propensity of DNA sequences for breakage.
Partial monosomy 21
We present eight new cases of partial monosomy (cases 33–36, 38–40 and 42) and a refinement of the mapping for three previously reported cases (31,40
). Cases 31 and 32, presenting a deletion of the proximal region of chromosome 21, were described with a severe phenotype including severe mental retardation, craniofacial abnormalities (broad forehead, downward-slanting palpebral fissures and low set, large ears). The 11 partial monosomies ranged in size from 1.48
Mb (case 42) to 21.06
Mb (case 31). All the partial monosomies were unique as none of the patients seems to share a common breakpoint (Figure 4). The smallest deletion (case 42) was estimated by aCGH to be only 1.48
Mb (Figure 4), and contains eight genes including DSCR1
. Case 42 has a relatively severe phenotype including mental retardation, microcephaly, short stature and cardiac anomaly.
The largest deletions mapped on chromosome 21 are 18.20
Mb (case 31) and 17.51
Mb (case 32) between the centromere and 21q22.11. Both of these cases have a severe phenotype including severe mental retardation, craniofacial abnormalities (broad forehead, downward-slanting palpebral fissures and low set, large ears). Cases 37 and 38 with distal deletions ranging from 11
Mb (case 35) to 5.63
Mb (case 37) have a relatively milder phenotype including moderate mental retardation and absence of craniofacial anomalies. Case 39 was reported with subtle dysmorphic anomalies including marfanoid habitus. The thin marfanoid build was previously associated with distal monosomy 21q.26, 43
The partial monosomy cases described here indicate how deletion of three broad regions of HSA21 contribute to the phenotype of monosomy. The first region, from the centromere to ~31.2
Mb produces a severe phenotype. This covers the gene-poor region of HSA21, but contains ~50 genes. In the second region from 31.2–36
Mb, there are no cases with a deletion spanning this region, and only one case (42) with a partial deletion of this region (case 42), and this case has a severe phenotype. This indicates that this region, which has a high gene density (~80 genes), contains a combination of genes that may not be tolerated in a monosomic state. The third region, from ~36–37.5
Mb to the telomere, contains a large number of genes ~130, but its monosomy results in a milder phenotype.
These data agree with previously published cases of partial trisomy 21.24, 27, 28, 29
For example, Chettouh et al24
presented six cases, four of whom had large proximal deletions and a severe phenotype, similar to cases 31 and 32 in our study. Also, Ehling et al27
presented two cases with distal deletions and a mild phenotype, consistent with cases presented here.
Given the rarity of partial monosomies, it is difficult to draw firm conclusions. However, for monosomy phenotypes, it seems that there is no region which would correspond to a ‘critical region' (). The cases presented here and in other studies24, 27, 28, 29
show that many of the individual phenotypes can be present when different regions of HSA21 are deleted.
Figure 3 Genotype–phenotype mapping in partial trisomy 21. Each graph represents one aspect of the phenotype (). The X-axis represents the position along HSA21q in Mb; Y-axis is the phenotype score for each BAC, with the maximal region shown in (more ...)
Partial trisomy 21 and critical regions
The 19 partial trisomies reported here range in size from 5.98
Mb (case 23) to 28.56
Mb (case 6), and each case is unique as none of the patients share a common breakpoint (). The clinical features of the cases included in this study were collected from the original medical records provided by the referring physicians. We evaluated the most frequent clinical feature described for the 19 cases of partial trisomies, and the available data and clinical evaluations are summarized in .
DS affects multiple systems and produces both functional and structural defects. T21 is frequently associated with mental retardation, congenital heart defects (mainly atrioventricular septal defect), abnormalities of the gastrointestinal tract, abnormalities of neuromuscular tone, characteristic facial and physical features, a high incidence of seizures, modified audiovestibular and visual functions and the early onset of Alzheimer's disease (AD). To date, almost every aspect of the phenotype of DS is subject to high degree of variability even in cases of full trisomy 21. Only two of these features are observed in all DS patients: mental retardation and neuropathological modifications similar to those observed in the brains of AD patients (in DS patients over the age of 35 years).
Typically, DS patients exhibit a progressive decline in IQ beginning in the first year of life. By adulthood, IQ is usually in moderate-to severe retardation ranged (IQ=25–55) with an upper limit on mental age of ~7–8 years, although a few individuals have IQ in the lower normal range (70–80 years).44
We report 13/16 (82%) cases with mental retardation in patient with partial trisomy 21, a lower rate compared to the value of 100% described in DS patients with full trisomy 21. The minimum region defined in our study maps is between 37.94 and 38.64 and contains KCNJ6
. However, cases 9, 10, 11 and 13 define a second region from the centromere to 26.96 contributing to MR.
Hypotonia, which is frequently observed in neonates, is difficult to associate with a well characterized developmental anomaly,45
but is reported as the most frequent sign of DS.1
In our study, hypotonia was present in 80% of the partial trisomy 21 cases, and enables the mapping of hypotonia region to two small genomic segments 37.4–38.4 and 46.5-qter.
The mapping of the triplicated genomic segment of chromosome 21 that harbors the functional elements contributing to congenital heart defect (CHD)46, 47
is of importance to understand the pathogenesis of these anomalies. CHD is present in only 2/12 (17%) compared to the overall risk of CHD of 40% reported in DS patients. Earlier studies of rare individuals with CHD and partial duplications of chromosome 21 established a candidate region from D21S3 to PFKL
which agrees with the two cases presented here (). However, the sample in our study maps the CHD region in a large genomic segment between 31.5
Mb and qter.
Earlier data show that not all the DS patient display microcephaly and the main feature observed is brachycephaly. Our study on partial trisomy 21 shows that 25 and 42% of partial trisomy 21 were reported with microcephaly and brachycephaly, respectively. Major craniofacial abnormalities well described in DS were reported with variable rate in our study; for example, upslanting palpebral fissure is present in 67% of the cases, flat facies in 34% and brushfield spots in 25% of the cases. shows the extent of the phenotypic mapping positions of these features.
In summary, partial trisomy patients display particular phenotypes at a lower frequency than trisomy 21 patients ().
The goal for mapping phenotypes to specific regions of HSA21 is to identify which genes (or small regions) contribute to DS phenotypic features, and thus to understand DS pathogenesis.48
Phenotype candidate regions are defined as the minimum region of overlap between cases positive for the phenotype. To map these regions, we calculated a score for each BAC along HSA21 (see Materials and methods for details). The scores are plotted in and , highlighting the highest scoring regions for each phenotype. Clearly, the majority of the phenotypes map to distal HSA21; averaging the phenotype score indicates a region ~37–44
Mb, which is involved in most DS phenotypes. This is not surprising, as this is the most gene-rich region of chromosome 21.49
Figure 4 Genotype–phenotype mapping in partial monosomy 21. Each graph represents one aspect of the phenotype (). The X-axis represents the position along HSA21q in Mb; Y-axis is the phenotype score for each BAC, with the maximal region shown in (more ...)
Confidence in these scores can be ascertained by assessing how many total cases express the phenotype compared to those with no phonotype (ie, penetrance; see the last column of ). Adjusting the scoring system described above for each BAC was done to take this type of data into consideration (see Materials and methods; Supplementary Figure S2). For example, cases 16 and 18 have cardiac anomaly, and the line () represents the region of overlap between the trisomic regions for these two cases (). However, in addition to these data, we have data from many more cases (8, 9, 10, 11, 12, 20, 23 and 25) that do not have cardiac anomaly, but are also trisomic for at least a part of this region. For many phenotypes, this analysis may reduce the size of the candidate regions, but it has the problem that it does not take into account the complicating factor of reduced penetrance (Supplementary Figure S2).
Cases 9, 10, 11, 12 and 13 are interesting as they are trisomic for proximal HSA21 and do not include the ‘DSCR' (). These cases, and three other similar cases reported previously,50
exclude the possibility of there being a single DSCR, in the sense of a single region being responsible for all aspects of the phenotype.
Although the most likely region for many phenotypes maps in 34–41
Mb, other regions are important for microcephaly, abnormal dermatoglyphics, short stature and furrowed tongue. Gene density correlates with DS (average) phenotype (Spearman rho 0.58, P
<0.0001), and this indicates that many genes along HSA21 make a contribution to the overall DS phenotype.
For a true DSCR, there must be no patients who have the major features of DS but do not have this region. Rather this is a susceptibility region (SR) modified by other loci on HSA21 and elsewhere in the genome. The DSCR may thus be described as a phenotype SR. These SRs make sense against a background of expression variation.51, 52
Mouse partial trisomies that are syntenic to trisomy 21 in humans provide important information regarding the genome mapping of phenotypic characteristics. For example, trisomy syntenic for the ‘DSCR' named Ts1Rhr11
did not display learning and memory abnormalities or facial dysmorphism, and thus did not provide evidence for the ‘DSCR'. However, genetic differences between strains and the problem of comparing phenotypes between mouse and human are an issue for DS mouse models in general. As in many other biological investigations, the positive result is significant, whereas the negative is uninformative.
Many more additional studies are needed to reduce the candidate regions for certain phenotypes. Given the rarity of these partial aneuploidies for chromosome 21, collection of cases from several centers need to be centralized and analyzed with a common diagnostic platform. In addition, a careful and structured evaluation of the phenotypic characteristics will result in meaningful comparisons and conclusions. The use of oligonucleotide platforms for array comparative genomic hybridization for diagnostic purposes may reveal a considerable number of additional cases that could otherwise be undiagnosed. There is a need for a web-based reporting and collection of both phenotypic and genotypic characterization.