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Editor—We report on a case with an apparent duplication of 22q with an initial karyotype of 46,XY,add(6)(p24).ish der(6)(6qter→6p24::22q11→22qter)(wcp6+,wcp22+). To our knowledge this is the first report of such an association. We have attempted to characterise the exact regions of duplication using fluorescence in situ hybridisation (FISH) techniques and confirmed that the case is indeed not deleted for chromosome 6 and is only partially trisomic for 22q, thus allowing a more detailed genotype-phenotype correlation than previous studies.
The majority of reports of duplication of chromosome 22 involve the proximal region on 22q. The most common cause of trisomy or tetrasomy of proximal 22q is a de novo supernumerary bisatellited marker chromosome derived from inverted duplication of 22p and 22q11. These supernumerary marker chromosomes have been associated with the cat eye syndrome (CES).1 In contrast, duplications of the distal part of the chromosome 22q region that arise de novo are relatively rare with only eight cases reported so far.2-9 A clinical picture of the distal 22q duplication syndrome has emerged from this limited number of cases comprising growth retardation, shortened life span, congenital heart defects, hypertelorism, narrow palpebral fissures, small nose with anteverted nares, small thorax with hypoplastic nipples, muscular hypotonia, feeding difficulties associated with failure to thrive, and anomalies of the external genitalia in males.5 9
The boy was born at 39 weeks after an uneventful pregnancy to healthy, non-consanguineous, 33 year old parents. It was the second pregnancy; the first pregnancy was spontaneously aborted at 6 weeks' gestation. Family history was normal. Clinical examination at birth showed a proportionately small infant; weight was 1500 g (−4.5 SD), length 39 cm (−5.9 SD), head circumference (OFC) 28.3 cm (−3.7 SD), and chest circumference 25.3 cm (−4.1 SD). He had cyanosis because of persistent fetal circulation, but congenital heart anomaly was not present. He had a round face, broad forehead, hypertelorism (not measured), low nasal bridge, depressed nasal tip, left unilateral cleft lip and cleft palate, low set and malformed ears, short neck, and low posterior hairline. There was no facial asymmetry. Hearing tests showed mild loss in both ears. Other anomalies included imperforate anus (ultrasonographic examination of the abdomen showed normal internal organs) and bilateral talipes.
Physical examination at 3 years 7 months showed poor weight gain, 9.36 kg (−3.1 SD), height 81.2 cm (−4.3 SD), and head circumference 42.8 cm (−4.1 SD). The patient had bilateral mild sensorineural deafness, 40-50 dB. Ophthalmological examination was performed and showed hypermetropic astigmatism and external squint. The optic field was normal. He showed moderate delay in psychomotor and growth retardation. He was able to control his head at the age of 6 months, sit independently at 1 year, and was able to walk without support at the age of 3 years 8 months. At 3 years he had no meaningful speech. The parents had apparently normal karyotypes. At this stage his molecular cytogenetic karyotype was 46,XY,add(6)(p24).ish der (6)(6qter→6p24:: 22q11→22qter)(wcp6+,wcp22+).
Cells were provided as an EBV transformed lymphoblast cell line. They were cultured at 37°C with 5% CO2 in RPMI 1640 medium containing 4 mmol/l L-glutamine, 10% FCS, 2% penicillin and streptomycin. Metaphase chromosome spreads were prepared using standard techniques. Briefly, actively growing cells were treated with thymidine at 280 μg/ml for approximately 18 hours. They were then washed in fresh medium and allowed to grow for five hours after which they were treated with colcemid solution for 15 minutes and then resuspended in 75 mmol/l KCl before being finally fixed in 3:1 methanol:acetic acid. The preparation was then used for FISH experiments.
Cosmid, YAC (total yeast DNA including YAC), PAC, and BAC DNA was prepared by standard techniques.10 All clones were labelled with biotin-14-dATP or digoxigenin-11-dUTP by nick translation (Bio-Nick Labeling System or Nick Translation System, respectively, BRL Life Technologies, USA). In situ hybridisation was performed as described previously.10 In total, 17 YAC, 16 PAC, 12 BAC clones, and one cosmid were used, of which the chromosome 6 clones had been previously FISH mapped to defined locations on 6p by us,11 while the chromosome 22 clones were part of the BAC clone set generated by Dr Nigel Carter at the Sanger Centre (http://www. sanger.ac.uk/HGP/Cytogenetics/Bacset.shtml) or contained physically mapped genetic markers (http://webace.sanger.ac.uk/cgi-bin/ace/simple/22ace) (table 1). The exact position of the chromosome 22 clones was retrospectively verified by the availability of the chromosome 22 sequence12 during the manuscript revision process (table 1).
Cytogenetic analysis of the case showed an abnormal short arm of one chromosome 6 carrying a deletion. Further FISH analysis was undertaken with 28 well characterised probes that covered the 6p24-pter region and a probe each for 6p23 and 6p21.2. All the probes hybridised with equal intensity to both normal and derivative chromosomes 6, indicating that the telomeric part of chromosome 6 is intact (table 1).
FISH with a whole chromosome 22 paint gave positive hybridisation signals along the entire length of both the normal chromosomes 22 as well as on the end of the derivative 6p arm. In order to establish which part of chromosome 22 is present in the derivative chromosome 6, we used 18 PAC and BAC clones with defined chromosomal locations or containing markers from the genetic map or both (table 1).
FISH with clone 213f7 located in 22q12.1-12.2 gave an additional signal on the end of the derivative 6p. Clones 115f6, 154h4, 433f6 (located in 22q11.2), 992d9 (located in 22q11.23), 99f11 (located in 22q12.1), 212a12, and 390b3 (located in 22q12.3-13.1) showed no additional signals on the short arm of chromosome 6, whereas clones 229a8, 394c4, and 722e9 located in 22q13.1→22q13.32 gave additional signals on the derivative chromosome (table 1). We observed the following order of hybridisation signals on the derivative chromosome: bk121L5 (6p21.2) - c6k23 (6p25.3) - dJ213f7 (22q12.1-12.2) - bk722e9 (22q13.31-13.32). Representative examples of results are shown in fig1.
In conclusion, we have established that the derivative chromosome 6 is not deleted for any region of 6p at least up to the subtelomeric marker D6S344 (the distance between cosmid 6k23 and D6S344 is not known). The rearranged duplicated chromosome 22 forms the telomeric portion of the derivative chromosome 6 (fig 2A). The duplicated regions include a small segment of the 22q12.1-q12.2 region between clones bk99f11 (not duplicated) and bk288l1 (not duplicated) (table 1, fig 2B). We can determine the maximum size of this duplicated segment, as detected by PAC dJ213F7 containing the marker D22S1175, from the available chromosome 22 data (http://webace.sanger.ac.uk/cgi-bin/ace/simple/22ace) as being 3.5 Mb. The 22q13 duplication starts from a region between the markers D22S344 (bk390b3, not duplicated) and D22S279 (bk229a8, duplicated) within q13.1 and extends through the rest of the long arm, covering a maximum of 14 Mb (table 1, fig2B).
Our results refined the cytogenetic diagnosis and enabled us to specify a new karyotype for the case (fig 2A): 46,XY,der(6)(6qter→6p24::22q11→22qter).ish der(6) (6qter → 6p25 :: 22q12.1 → 22q12.2 :: 22q13.1→ 22qter) (bk121l5+, 912g9+, 886a2+, 844h3+, 915e8+, 938d8+, 436h5+, 753a9+, 886c1+, dJ133h11+, dJ135k15+, dJ110p13+, dJ167k8+, dJ108c15+, dJ359l13+, bk3J9+, dJ103m22+, 808a10+, 848c12+, 963h4+, 786h6+, 938b10+, 863b6+, 878b10+, dJ36i2+, 954h10+, 947d4+, c6k23+, bk722e9+, dJ127b20+, bk397c4+, dJ186O1+, bk229a8+, dJ124O7, bk212a12−, bk390b3−, dJ78b3−, dJ153h10−, dJ288l1−, dJ213F7+, bk99f11−, dJ90l6−, bk992d9−, bk433f6−, bk154h4−, bk115f6−).
The FISH with well defined probes confirmed that this case is partially trisomic for 22q12.1-12.2 and 22q13.1-qter without any deletion of 6p. This anomaly is not a result of malsegregation states since both parents have a normal karyotype.
Although the chromosomal rearrangement of our case is unique, the most cytogenetically similar case published to date is the one by Wieczoreket al.9 Although Wieczoreket al 9 did not perform a detailed FISH analysis, comparison of the markers contained in the probes used show that the reported 22q13 duplication has occurred distal to the marker D22S102. The breakpoint was not identified since the next marker used in their study is close to the telomere (ARSA13). The breakpoint in our case occurred proximal to D22S279 and distal to D22S344 and D22S156 (and therefore also distal to D22S102) within a 2 cM interval.
The phenotypes are comparable, namely protuding forehead, hypertelorism, broad nasal bridge, orofacial clefting, low set ears, growth retardation, and psychomoter retardation. In contrast to their case, our case also had imperforate anus, hearing loss, and talipes. Our results clearly reinforce the findings associated with distal 22q duplication and the correlation of the phenotypic characteristics with the duplication of 22q13.1-qter is now confirmed by molecular techniques. Table 2 summarises the phenotypic comparison between this case, Wieczorek et al,9 and the review of previous cases in Wiezcorek et al.9
Anal atresia has rarely been observed in cases presenting with proximal or distal interstitial duplications and it is often associated with many other malformations. The anomaly is mainly associated with CES, derivative (22) syndrome, or complete trisomy.14-17 The CES critical region has been defined as an approximately 2 Mb segment between the centromere and marker D22S57.18 A part of this region has been cloned19 and two of the BAC clones used in this study (bk115f6 and bk154h4) are also part of the contig presented by Johnson et al,19 thus enabling a direct comparison with their data. The proximal breakpoint of the proximal duplication in the case described here is approximately 9 Mb distal to the distal breakpoint cluster identified in CES patients.20 Therefore, unless an additional undetected part of chromosome 22 is duplicated in the 1.7 Mb region between the centromere and bk115f6, we can conclude that the duplications identified in this case are unrelated to the CES critical region (fig2B). Our case may be significant in that it proposes that overexpression of 22q12.1→22q12.2 distal to the regions associated with CES or of 22q13 may be involved in the pathogenesis of anal anomalies. Alternatively there could be a duplication of a small segment of 22q11 that lies between the PAC and BAC clones used and remains undetected or there is an undetected deletion of 6p near the telomere that may influence the phenotype. Position effects on the expression of genes within 6p25 are also a possibility. However, anal abnormalities are not part of the distal 6p deletion syndrome.11 The breakpoints of our case are distributed on the q arm, the proximal breakpoints being near 22q12.1-22q12.2 and the distal breakpoint in 22q13.1. There seems to be a correlation between the position of the common aphidicolin inducible fragile site found at 22q12.2 and our proximal breakpoints and the rare folate sensitive fragile site FRA22A, recognised at 22q13, with our distal breakpoint.21
We have shown by using individual clones that we were able to define clearly the nature of the anomaly and delineate and segregate the clinical presentations associated with specific segments. The conduct of similarly detailed studies will allow more precise comparisons between individual cases in the future.
We would like to thank the UK's HGMP-Resource Centre for providing PAC clones, Dr Ian Dunham (Sanger Centre) for providing BAC clones, and Dr Angela Davies and Dr Paul Scriven for discussion.The work was supported by MRC grant G9533412.
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