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Partial tetrasomy 21 in a male infant
  1. ANNE M SLAVOTINEK*,
  2. XIAO-NING CHEN,
  3. ANN JACKSON*,
  4. LORRAINE GAUNT*,
  5. ALASTAIR CAMPBELL,
  6. JILL CLAYTON-SMITH*,
  7. JULIE R KORENBERG
  1. *University Department of Medical Genetics, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK
  2. Medical Genetics Birth Defects Center, Cedars-Sinai Medical Center, UCLA, Los Angeles, CA, USA
  3. Department of Paediatrics, Royal Preston Hospital, Preston PR2 9HT, UK
  1. Dr Slavotinek, National Human Genome Research Institute, National Institutes of Health, Room 4B75, Bldg 49, 49 Convent Drive, Bethesda MD 20892, USA, aslavoti{at}nhgri.nih.gov

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Editor—Tetrasomy 21 without mosaicism has previously been described in four liveborn children, two of whom had physical features consistent with Down syndrome. We describe a male infant with partial tetrasomy 21 whose examination was consistent with but not typical of trisomy 21.

The clinical features of the proband are summarised in table 1. The proband was the second child born to healthy white parents. Their first child was a normal son aged 18 months. There was no significant family history and no consanguinity. The mother was 34 years old at the time of the birth and the father was 33 years old.

Table 1

Summary of the physical characteristics of the proband

During the pregnancy poor weight gain was reported, although the mother stated that the fetal movements were normal. A detailed ultrasound scan at 18 weeks of gestation was normal. A repeat scan at 30 weeks of gestation showed intrauterine growth retardation.

Labour occurred spontaneously at 33 weeks of gestation and an emergency caesarian section was performed for fetal distress and growth retardation. The birth weight was 1420 g (3rd centile), length 38 cm (1.8 cm less than the 3rd centile), and head circumference 27 cm (0.4th centile). The proband required special care because of his prematurity and small size. Hypospadias, a bifid scrotum, and microcephaly were noted and blood was sent for chromosome analysis on the second day of life (see below). The proband was tube fed for the first 4 weeks of life because of nasal regurgitation and difficulty coordinating sucking and breathing. There were no reports of neonatal hypotonia. A small umbilical hernia was noticed at 1 week of age. He was discharged at 6 weeks of age feeding well with a weight of 2000 g (200 g less than 0.4th centile).

On examination at 5 months (fig 1), length was 55 cm (just below the 0.4th centile), weight 5.8 kg (25-50th centile), and head circumference 37 cm (1 cm less than the 0.4th centile). The anterior fontanelle was small. He had a high anterior hairline with a widow's peak and small, delicate eyebrows, different from other family members. There was bilateral ptosis and he held his head back to improve his vision. There were small epicanthic folds and he had hypertelorism (table 2), but his irides appeared normal. The nasal bridge was wide and he had a short, grooved philtrum with a small, triangular shaped mouth and a small chin. His ears were normally sited and had pointed helices. The tongue was large but the palate was normal. He had a short neck with redundant folds of skin. His hands and feet were small with brachydactyly of the digits and short, proximally inserted thumbs. The palmar and digital flexion creases were normal. There were two 1-2 cm strawberry naevi on the posterior aspect of the right scalp and on the right shoulder. There was a large, soft, reducible umbilical hernia. His penis was small with hypospadias and a shawl scrotum. Both testes were descended. He had mild truncal hypotonia. Cardiac auscultation was normal.

Figure 1

(A) Face of the proband aged 5 months. (B) The body of the proband.

Table 2

Summary of the growth characteristics of the proband

He has had frequent “absences” thought to be seizures since the neonatal period. An EEG has been normal and trial of medications has not been undertaken. A CT scan of his head at 4 months showed cerebral hypodevelopment, the degree of which was greater than expected for corrected age, but there were no structural lesions.

At 18 months (fig 2), he had good head control and was able to roll over and to babble. He had plagiocephaly and there was marked brachycephaly with a flat, underdeveloped midface. His hypertelorism and ptosis were unchanged and there was a left divergent squint. His tongue remained large and protruded from his mouth. His fifth toenails were hypoplastic. The remainder of the examination was unchanged. At 34 months of age, he had been sitting independently for 1 month, weight bearing for several months, and could understand his name but no other words. His tone was normal and there were no epicanthic folds or redundant neck skin. He had seven teeth. Investigations have included a normal renal ultrasound scan and electrocardiogram but echocardiography has not been performed.

Figure 2

(A) Face of the proband aged 18 months. (B) The body of the proband. (C) Side view of proband. (D) The hand of the proband. (E) The foot of the proband.

Chromosome analysis of peripheral blood lymphocytes showed a small supernumerary chromosome in all cells (fig 3A, B). Fluorescence in situ hybridisation (FISH) studies with a whole chromosome paint for chromosome 21 (Cambio) showed that the extra material was derived from chromosome 21 (fig 4A) and a 13/21 centromere probe (Oncor) showed that the derivative chromosome was an isodicentric chromosome 21 (fig 4B). The proband was therefore tetrasomic for the proximal part of the long arm of chromosome 21 to band 21q22.1 (G banded karyotype 47,XY,+psu idic(21)(q22.1)). Parental chromosomes were normal. Repeat cytogenetic analysis at 18 months showed that the isodicentric chromosome 21 was present in 30/30 peripheral blood lymphocytes and in 30/30 skin fibroblasts. FISH studies on peripheral blood lymphocytes showed no signal from the cosmid clones ICRFc102E0275, ICRFc102H01108, and ICRFc102F01129 mapped to chromosome band 21q22.3 (fig 4C, Imperial Cancer Research Fund Reference Library Database, Lincoln's Inn Fields, London WC2A 3PX).1 2 The duplicated region in this child therefore did not include the Down syndrome critical region.

Figure 3

(A) Partial G banded karyotype of the proband, showing small, supernumerary chromosome. (B) G banded examples of the pseudoisodicentric chromosome 21.

Figure 4

(A) FISH study with a whole chromosome painting probe for chromosome 21 showing that the supernumerary chromosome is derived from chromosome 21. (B) FISH study with a centromere probe for chromosomes 13 and 21 showing two signals on the supernumerary chromosome. (C) FISH study with cosmid clones ICRFc102E0275, ICRFc102H01108, and ICRFc102F01129 (see text) showing absent signals on the supernumerary chromosome. (D) A human chromosome preparation made from the proband hybridised with a Cy3-dUTP direct labelled probe, B839E9. Four copies (shown in red) are present in both metaphase and interphase cells.

To determine the extent of the tetrasomic region, a total of 12 DNA probes were tested using FISH analysis (table 3). These probes included four cosmids and eight BACs. All of the BAC probes were obtained by screening a BAC library (A and B)3 4 using DNA from chromosome 21 as a probe5 or from the Integrated Molecular Cytogenetic BAC Resource.6 Their chromosome locations were confirmed by testing on normal control metaphase chromosomes and by using PCR with custom designed primers as indicated in table 3.

Table 3

DNA probes used for characterisation of the tetrasomic region of chromosome 21

BAC and cosmid DNA preparations, probe labellings, and FISH were performed according to the procedure described by Korenberget al 6 with some modification. Briefly, 10 μl of hybridisation mix (50% deionised formamide, 10% dextran sulphate, and 2 × SSC) containing 200 ng of each Cy3-dUTP or Cy5-dUTP (Amersham Life Science) directly labelled DNA probes, 5 μg of human Cot1 DNA, and 5 μg of sonicated salmon sperm DNA was applied to denatured chromosomes after preannealing the probe for 20 minutes at 37°C. Post-hybridisation washes were at 44°C in 2 × SSC and 50% formamide for five minutes three times and followed by washing once in 1 × SSC at 50°C. Chromosomes were identified by reverse banding generated with chromomycin A3 followed by distamycin A5and the hybridisation signals were directly reviewed after the counterstain. The images were captured with a Photometrics cooled CCD camera mounted on Zeiss Axiovert 135 microscope using BDS image software (Oncor Imaging, Gaithersburg, MD).

Characterisation of the pseudoisodicentric chromosome 21 of the proband showed that the duplicated segment extended to the distal region of band 21q21, excluding the genes for APP,GRIK, and SOD(fig 5). The distal breakpoint was located between BAC 839E9 showing four copies (fig 4D) and 427H6 showing two copies. The three copies of probe E9 suggest that this probe maps close to or at the proximal breakpoint of the isodicentric chromosome. BAC 839E9 corresponds to YAC 221B7, carrying the markers D21S383, D21S384, and D21S385 and BAC 427H6 is positive for YAC 193G12 and carries markers D21S18 to D21S281. The duplicated region therefore covers the region from D21S16 to D21S1/S11 and possibly extends to D21S281. The distal breakpoint of the isodicentric chromosome characterised by FISH revises the karyotype to 47,XY,+psu idic(21)(q21.1).

Figure 5

Molecular cytogenetic analysis with BAC and cosmid probes. A human chromosome 21 ideogram showing the duplicated region and genes. The probes tested are indicated with *.

We have reported a 34 month old boy with tetrasomy for the short and proximal long arm of chromosome 21 to band 21q21.16 without evidence of mosaicism in peripheral blood lymphocytes and skin fibroblasts. His phenotype was suggestive of but not entirely consistent with Down syndrome. Brachycephaly, epicanthic folds, a flat nasal bridge, an open mouth and protruding tongue, short neck with excess skin, small hands and feet, fifth finger brachydactyly and clinodactyly, and an umbilical hernia gave a Jackson score of 12/25 at 18 months of age, consistent with an 84% probability of Down syndrome (table 4).7However, his examination at 5 months of age showed fewer signs of Down syndrome and he had a high anterior hairline, non-slanting palpebral fissures, bilateral ptosis, and a triangular shaped mouth (fig 1). At 34 months of age, epicanthic folds and redundant neck skin were absent and his tone was normal. His Jackson score at this age was 9/25.

Table 4

Clinical features of cases with tetrasomy 21 and tetrasomy 21 mosaicism

Tetrasomy for an autosomal chromosome region has been described for only a few chromosomes.8 Autosomal tetrasomies are almost always de novo aberrations, with tetrasomy resulting from an additional isochromosome or isodicentric chromosome. More rarely, two extra copies of an acrocentric chromosome may be joined in a Robertsonian translocation or there can be two supernumerary whole chromosomes. Mosaicism is frequent and can result from mitotic instability of the isodicentric chromosome, in vivo growth disadvantage of the aberrant chromosome(s), or failure of the tetrasomic cells to divide in vitro.8 9

Partial tetrasomy 21 without mosaicism has previously been described in four liveborn infants (tables 4 and 5). A 33 month old girl with tetrasomy extending to chromosome band 21q22.1 had brachycephaly, a third fontanelle, short, upward slanting palpebral fissures, and a protruding tongue. Single transverse palmar creases, brachydactyly, an umbilical hernia, joint laxity, and hypotonia were also noted (table4).10 A baby born at 32 weeks had congenital monocytic leukaemia and two additional copies of the whole of chromosome 21 in peripheral blood lymphocytes.11 No karyotyping was performed on skin or bone marrow cells. The child had a flat occiput, small, upward slanting palpebral fissures, epicanthic folds, small ears with unfolded helices, a flat nasal bridge, a short neck with redundant skin, and marked hypotonia consistent with Down syndrome. Death occurred at 4 days of age from hyaline membrane disease and septicaemia and necropsy showed a patent ductus arteriosus and a bilobed right lung.11 Recently, a male aged 30 months was reported with a pseudoisodicentric chromosome 21.12 He had developmental delay, microcephaly, hypotonia, joint laxity, a flat occiput, downward slanting palpebral fissures, Brushfield spots, telecanthus, and midface hypoplasia.12 The authors concluded that the child had some but not all of the features of Down syndrome and that genes outside the Down syndrome critical region (DCR) were influential in the trisomy 21 phenotype.12 Tetrasomy 21 was also described in an 11 year old male with psychomotor and speech retardation (but with normal growth), brachycephaly, a prominent lower lip, macrogenitalism, and idiopathic precocious puberty.13 The Jackson score for this child was not consistent with Down syndrome.13

Table 5

Karyotypes of cases with tetrasomy 21 and tetrasomy 21 mosaicism

There are five reports of tetrasomy 21 in mosaic form.14-18 However, several of these cases were reported before confirmatory FISH studies were available, and one case15 has subsequently been shown to have Pallister-Killian syndrome with mosaic tetrasomy for chromosome 12p rather than for 21q.19 The diagnosis in three of the other cases14 16 17 has also been challenged in view of the phenotypic similarity of these cases to Pallister-Killian syndrome.20 21 One case of partial tetrasomy 21 in mosaic form verified by FISH had some features consistent with Down syndrome (table 4) with increased nuchal skin, mild brachycephaly, Brushfield spots, a right epicanthic fold, a depressed nasal bridge, bilateral fifth finger clinodactyly, and a wide sandal gap.18

Our case provides additional evidence that the phenotype associated with non-mosaic tetrasomy 21 may include physical features uncommon in trisomy 21. Findings in previously reported cases of both mosaic and non-mosaic tetrasomy 21 include a flat occiput,10 (our patient) a high, broad forehead,12 18 (our patient), hypertelorism or telecanthus,12 (our patient), and a short and anteverted nose13 (our patient). Our patient also had a carp shaped mouth. Non-specific genitourinary anomalies were more frequent (table 4), and cryptorchidism,11 12 hypospadias, and a small scrotum (our patient) and a large penis13 have been reported. A patent ductus arteriosus was described in one premature infant,11 but no other case had a cardiac abnormality. Two cases had an umbilical hernia10 (our patient) and one had diastasis recti,10 but there were no other gastrointestinal abnormalities. One child had pectus excavatum.12 It seems likely that the phenotype associated with tetrasomy 21 will prove to contain features distinct from Down syndrome, although the few cases described to date and the short time period of follow up do not yet allow characterisation of a tetrasomy 21 phenotype.

The phenotypic effects of four copies of an autosomal chromosome region are likely to be more severe than for trisomy of the same chromosome segment as a result of a correspondingly greater imbalance in gene dosage.7 22 23 This hypothesis holds true for the majority of cases of tetrasomy 21 described to date, as development and growth were more severely impaired than would have been expected with trisomy 21 (table 4). However, there was considerable variation in both growth and development and one girl with non-mosaic tetrasomy 21 walked independently at the age of 18 months and had single words at the age of 33 months.10 A child with normal growth parameters13 and a child with normal height and weight but with microcephaly have also been reported.10

Molecular cytogenetic characterisation of the tetrasomic region in this patient showed that the duplicated region covers the region from D21S16 to D21S1/S11. This region contains only two known genes,STCH and RIP140. STCH represents the stress-70 chaperone family, consisting of proteins that bind to denatured or incorrectly folded polypeptides and play a major role in the processing of cytosolic and secretory proteins.24 RIP140 represents the receptor interacting protein 140 by virtue of its direct association with a transcriptional activation domain of the oestrogen receptor (ESR, 133430) in the presence of oestrogen.25 Therefore it is likely that some of the physical features and delayed development derive from the overexpression of these genes during development. It is also of interest that subjects who are neither monosomic nor partially trisomic for these regions exhibit such abnormal phenotypes. It will be of interest to determine if more general effects on transcription may result from the overexpression of regulators such asRPI140.

Cytogenetic and molecular studies have been used to delineate specific regions of chromosome 21 responsible for the pathogenesis of different features in Down syndrome. Reports of subjects with partial duplications of chromosome 21 and a subset of the physical manifestations of trisomy 21 have identified a critical region for the phenotype at distal 21q21.3 to 21q22.26-28 Further molecular studies have identified a 5 Mb region between markers D21S58 and D21S42 with duplication of this region sufficient to cause the facial features and mental retardation associated with trisomy 21.29-33 However, duplications of chromosome 21 distinct from this region also produce phenotypic features of Down syndrome and it is likely that there is a significant contribution from other genes on chromosome 21 and elsewhere in the genome.12 33 34Our case is in keeping with the involvement of genes outside this “critical” region in the trisomy 21 phenotype as the tetrasomic region in our patient does not include this chromosome region and yet examination showed many features in accordance with Down syndrome.

Tetrasomy 21 has been well documented as an acquired cytogenetic aberration in subjects with a normal constitutional karyotype who have developed haematological malignancies. The conditions associated with tetrasomy 21 include acute lymphoblastic leukaemia (ALL), acute megakaryoblastic leukaemia (AML), erythroleukaemia, and transient myeloproliferative disorders.35-37 Children with Down syndrome are also well known to be at increased risk of leukaemia, most specifically ALL and AML.31 The link between the additional copies of chromosome 21 and haematological malignancy is therefore undoubted, although the pathogenesis of the malignant proliferation is still unknown. A role has been suggested for overexpression of the human ets-2 anderg proto-oncogenes mapped to 21q22.2 to 21q22.3.38 In tetrasomy 21, the one child described with four whole copies of chromosome 21 in peripheral blood lymphocytes (thus including the region containing the proto-oncogenes) was born with monocytic leukaemia.11 The other surviving cases either had mosaicism largely confined to skin fibroblasts or did not have additional copies of the 21q22.2 to 21q22.3 region (table 5).

Finally, in our case, the pseudoisodicentric chromosome contained material from both the short and the long arm of chromosome 21 and could have been formed from a U loop mechanism.39 In the surviving cases of tetrasomy 21 with mosaicism, the aberrant cells are almost always confined to the fibroblast cell lineages and are absent from peripheral blood lymphocytes (table 5). This is similar to the pattern of mosaicism in tetrasomy 12p40 and is thought to be because of selective loss of the isochromosome during cell division in vitro.9

We report a 34 month old child with tetrasomy for the short and long arm of chromosome 21 to band 21q22.1 determined cytogenetically, but only to 21q21.1 as determined by FISH analysis. There was no evidence of mosaicism in peripheral blood lymphocytes and skin fibroblasts. His facial features of brachycephaly, a high and broad forehead, epicanthic folds, bilateral ptosis, hypertelorism, a flat nasal bridge and small nose with anteverted nares, a small mouth, protruding tongue, and a small chin were consistent with but not classically in keeping with the trisomy 21 phenotype. A short neck with loose skin, brachydactyly with fifth finger clinodactyly, umbilical hernia, and small hands and feet were also present. A review of previously reported cases with non-mosaic tetrasomy 21 and mosaicism for tetrasomy 21 suggests that the phenotype for tetrasomy 21 will prove to contain features distinct from Down syndrome.

Acknowledgments

We are grateful to Jonathan Dore for his kind help with the illustrations. This work was supported in part by NIH grants R01 HL50025 (JRK), P50 HL61033 (JRK), and P01 HD17449 (JRK).

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