Mosaic Supernumerary Ring Chromosome 19 J Med Genet: First Published As 10.1136/Jmg.35.10.836 on 1 October 1998

83686Med Genet 1998;35:836-840

Mosaic supernumerary ring chromosome 19 J Med Genet: first published as 10.1136/jmg.35.10.836 on 1 October 1998. Downloaded from identified by comparative genomic hybridisation

Saeed R Ghaffari, Elizabeth Boyd, J Michael Connor, Alison M Jones, J L Tolmie

Abstract

We report the use of comparative genomic hybridisation (CGH) to define the origin of a supernumerary ring chromosome which conventional cytogenetic banding and fluorescence in situ hybridisation (FISH) methods had failed to identify. Targeted FISH using whole chromosome 19 library arm and site specific probes then confirmed the CGH results. This study shows the feasibility of using CGH for the identification of supernumerary marker chromosomes, even in fewer than

50% of cells, where no clinical or cyto- .i

genetic clues are present. (JMed Genet 1998;35:836-840) Keywords: chromosome 19; comparative genomic hybridisation; ring chromosome; mosaicism Figure 1 The proband. The origin of supernumerary marker chromo- complementary experiments by FISH con- somes cannot always be resolved by means of firmed the CGH results. conventional banding techniques.' When there are cytogenetic or clinical clues to the chromo- Materials and methods some abnormality, FISH using appropriate CASE REPORT The proband (fig 1), a 71 year old woman with probes may identify the origin of the marker http://jmg.bmj.com/ chromosome, otherwise a stepwise approach mental handicap, had been in residential care using different FISH probes or reverse chro- for 55 years. No details of her birth history or mosome painting,2-5 possibly combined with antenatal life were available. The family chromosome microdissection, is required."' recalled her walking at the age of 2 years and chromosome is the development of speech was delayed until However, when the marker the age of 4 or 5 years. She was not clumsy but very small, or has no significant band land- always had some difficulty with fine motor mark, or is present in a low percentage of cells skills. She attended a private convent school in on September 26, 2021 by guest. Protected copyright. only, even FISH may fail to identify the abnor- early childhood but upon her mother's death, mality. Both FISH and microdissection also when the proband was aged 16 years, she was depend on good quality metaphases with high received into care and has resided in institu- mitotic index, conditions that may be difficult tions ever since. The proband was the ninth to obtain. Therefore, a genome wide screening child born in a family of 10. In the family, no technique is desirable. sib was similarly affected to the proband and Comparative genomic hybridisation (CGH) there was no history of mental or physical Institute ofMedical is a genome wide screening technique that was illness. In 1976 the proband was admitted to Genetics, Yorkhill first developed to study chromosome abnor- Hospitals Campus, hospital for treatment for hypertension and Yorkhill, Glasgow malities in tumours. 1-13 It has also been used as impaired renal function of unknown origin. At G3 8SJ, UK an adjunct to conventional cytogenetics for the that time, she also had septic skin lesions and S R Ghaffari diagnosis of chromosome imbalance in clinical osteoarthritis affecting the hips. At present, she E Boyd cytogenetics."'6 However, there are no reports is able to feed, wash, and dress herself (with J M Connor of CGH being used as a tool to diagnose the some assistance), handle toys, obey simple J L Tolmie origin of the extra DNA material in patients commands, and guard against physical dan- Merchiston Hospital, who have low percentage mosaicism for a gers. Speech is mostly indistinct but she can Brookfield, By small, supernumerary, marker chromosome. utter short simple sentences that convey mean- Johnstone PA5 8TY, Here we report a 71 year old woman with ing. She is able to write a few words and can UK mental retardation and a supernumerary also read several words, but is notably skilled at A M Jones marker chromosome, the genetic origin of completing jigsaw puzzles. It is thought osteo- Correspondence to: which was not detected despite attempts using arthritis has led to loss of some fine motor Dr Boyd. conventional cytogenetics and, latterly, FISH skills. The proband has never been independ- using centromeric probes for a number of ent or able to go out on her own but she enjoys Received 13 November 1997 She has and bowel conti- Revised version accepted for chromosomes. In contrast, CGH readily social events. urinary publication 13 March 1998 showed its origin from chromosome 19 and nence. Mosaic supernumerary ring chromosome 19 837

Physical examination showed a height of 132 tific, Cambridge, UK). Green, red, and blue cm, head circumference of 52.5 cm, blood fluorescence images were captured from each J Med Genet: first published as 10.1136/jmg.35.10.836 on 1 October 1998. Downloaded from pressure of 180/110 mm Hg, poor peripheral high intensity, uniformly hybridised metaphase circulation, early hypertensive changes in both and were analysed as separate grey scale optic fundi, poor vision, upward slanting images. The image representing the blue DAPI palpebral fissures, large tongue, protruding counterstain was inverted and used for chro- lower lip, normal palmar creases, normal mosome identification based on its coarse female genitalia, and deep set toe nails. She has banding pattern. The mean of the individual reduced muscle power, a shuffling gait, bilat- ratio profiles of 20 metaphase spreads was cal- eral pes planus, and radiological signs of culated. For normalisation of the ratio profiles, subluxed, osteoarthritic hip joints. An x ray in the model value ofthe green to red ratio for the 1994 was reported to show considerable entire metaphase was set to 1.0. Finally, the degenerative changes in the cervical spine with individual ratio profiles were displayed next to gross osteophyte formation and partial, pre- the chromosome diagrams. sumed congenital, fusion of vertebral bodies C3 and C4. Recently, renal function has FLUORESCENCE IN SITU HYBRIDISATION declined with recurrent urinary infections and In an earlier attempt to identify the supernu- her right kidney is non-functioning. merary chromosome, FISH to metaphase Detailed psychological examinations were chromosomes was carried out using a number performed and the highest estimate of intellect of commercially available centromeric probes suggested moderate to mild learning disability. (chromosomes 4, 12, 20, 22, 13/21, and However, the British Picture Vocabulary Scale 1/5/19). After the CGH results were obtained, result and the examiners' impressions indi- targeted FISH was carried out using whole cated that moderate to severe learning disabil- chromosome 19 paint (Oncor), 19 centromeric ity was present. Notably, although there was probe, 19p and 19q arm paints, and telomere some evidence of behavioural and intellectual specific probes for l9p and 19q(LI), according deterioration, there were no psychological or to the standard FISH procedures. psychiatric difficulties in an emotionally stable woman who has good interpersonal relation- Results ships. CONVENTIONAL CYTOGENETIC ANALYSIS (FIG 2A) In the proband's residence, it was assumed Cytogenetic studies by G banding in 1976 that she was affected by Down's syndrome but showed the presence of two cell lines. Eighteen the true karyotypic abnormality was discovered out of 25 cells showed a 46,XX karyotype while in 1976 when a blood sample was first submit- seven out of 25 (28%) showed an additional ted for chromosomal analysis. marker chromosome of unknown origin (46,XX/47,XX,+marker). Chromosome CHROMOSOME PREPARATIONS analysis by G banding in 1986 confirmed the Metaphase spreads were prepared from phyto- finding of two cell lines. Twenty-one out of 25 http://jmg.bmj.com/ haemagglutinin (PHA) stimulated peripheral cells examined showed an apparently normal blood lymphocytes ofthe patient (for FISH) or female karyotype while the remaining four cells healthy males (for CGH studies) using stand- (16%) showed a count of 47 including one ard procedures of hypotonic treatment and additional small chromosome. This marker methanol/acetic acid fixation (3:1 v/v). appeared to be a ring chromosome, approxi- mately halfthe size of a chromosome 21, which CGH AND DIGITAL IMAGE ANALYSIS contained at least one G band positive area (fig on September 26, 2021 by guest. Protected copyright. Genomic DNA probe preparation, labelling 2A). C banding showed one centromere to be procedures, hybridisation, and posthybridisa- present, and Ag NOR staining was negative tion washings were carried out as described with respect to the ring chromosome. No other previously.'6 Briefly, test and control DNA was members of the family underwent cytogenetic extracted by proteinase K and RNase diges- investigation. tions. Control genomic DNA was prepared from blood of healthy females (46,XX). Test FIRST SERIES OF FISH STUDIES (patient) human genomic DNA was directly FISH was carried out using centromeric labelled with FITC-12-dUTP (DuPont) and probes for chromosomes 4, 12, 20, 22, 13/21, control DNA was labelled with Texas Red-5- and 1/5/19 empirically. The studies failed to dUTP (DuPont) by standard nick translation detect the origin of the extra chromosome and reaction; DNase I concentration was adjusted no further probes were investigated because to result in an average fragment size of difficulty was experienced in identifying the 500-1000 bp. One microgram of fluorescein marker with certainty within the metaphases. labelled test DNA, 1 jg of Texas Red labelled reference, and 50 gg of unlabelled human CGH ANALYSIS (FIG 2B) Cot-i DNA (Gibco) were cohybridised to nor- CGH was carried out using a DNA sample mal female metaphase spreads. Hybridisation from the patient. Analysis using a very restric- was allowed to proceed for 48 hours at 37°C in tive threshold with a fluorescence ratio of 0.5- a moist chamber. After posthybridisation 1.5 (which requires 100% abnormal cells to washings, images for CGH analysis were detect any gain or loss of the DNA material) acquired using an epifluorescence microscope failed to detect any abnormality. Progressively (Axioplan, Zeiss, Germany) equipped with a less restrictive thresholds were then used and, cooled CCD camera (Photometrics) control- surprisingly, a threshold of 0.8-1.20 (which led by an image analysis system (Digital Scien- theoretically detects abnormal cells comprising 838 Ghaffari, Boyd, Connor, et al

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Figure 2 Conventional cytogenetic, CGH, and targeted FISH results. (A) G banded metaphase spread of the patient showing a supernumerary ring chromosome (arrowed). (B) Standard ratio profile ofchromosome 19 is shown above the chromosome idiogram. The horizontal lines represent the balanced state of the chromosomal copy number (middle line, ratio 1. 0) and the lower and upper thresholds applied to detect chromosomal losses and gains, respectively. These thresholds correspond to ratios of 0. 8 and 1.20, which are the theoretical values expectedfor a monosomy or trisomy present in 40% of diploid cells. (C, D) FISH to metaphase chromosome from the patient using chromosome 19p (C) and 1 9q arm specific painting probes (D). 40% of the total) showed a gain in DNA mate- case since in each instance non-homologous

rial corresponding to 1 9q (fig 2B). In CGH the chromosomes were involved and the patterns on September 26, 2021 by guest. Protected copyright. centromeric areas are strongly suppressed by of clinical abnormalities which resulted were unlabelled human Cot-i DNA, so gain or loss quite variable. of this area could not be interpreted reliably. Among the four cases with supernumerary ring 19, one, a young infant with macrocephaly FISH CONFIRMATION OF THE CGH RESULTS (FIG and developmental delay, is not directly 2C, D) comparable as metaphase cells in that case Further FISH studies using a whole chromo- contained dicentric marker chromosome 19 as some 19 paint (WCP19, Oncor), 19p and 19q well as another marker chromosome, not 19.2 arm specific paints, and telomeric probes for Among the remaining three cases, the first 19p and 19q (LI) and 19 centromeric probe case2 was a newborn infant aged 3 weeks with confirmed the CGH finding. The ring chromo- failure to thrive, hypotonia, and pneumonia some hybridised to the whole chromosome 19 with a marker chromosome 19 in 50% of the and 19q arm specific paints and chromosome peripheral lymphocytes examined. The third 19 centromeric probe but not to l9p arm spe- case with supernumerary r(19)" had a very cific paint or l9p or 19q telomeric probes. It small supernumerary r(19) present in 46% of was thus concluded that the proband was umbilical cord blood cells and estimated to be trisomic for almost the entire long arm of chro- about 1/6 the size of chromosome 21. This was mosome 19 with only the telomeric region identified after amniocentesis was performed being absent from the ring. for advanced maternal age. The normal male infant weighed 3700 g at birth and he had nor- Discussion mal growth and development when evaluated To date, only four cases with a supernumerary aged 18 months. In another case, Quack et al17 ring 19 have been reported.2 17 18 Although reported a non-mosaic, supernumerary r(19) there are 11 reported cases with partial 19q present in a boy with somatic overgrowth and trisomy,9 26 none is directly comparable to our mental retardation. Unusually, this boy's Mosaic supernumerary ring chromosome 19 839

mother carried the same ring 19 but had a bal- rescence ratio of 0.75-1.25, there should be at anced karyotype since one other chromosome least 50% abnormal cells present for the chro- J Med Genet: first published as 10.1136/jmg.35.10.836 on 1 October 1998. Downloaded from 19 had an interstitial deletion of the long arm mosome imbalance to be detected.'7-29 In the segment of chromosome 19 which formed the present case, fluorescence ratio 0.8-1.20 was ring. Thus, the ring chromosome was shown to the most restrictive threshold at which the be derived from the deleted chromosome after abnormality was detected, which leads to an the occurrence of two breaks, one in the estimation of the percentage of abnormal cells centromere region, the other in band q13.2 in in uncultured blood of at least 40%. This pro- the long arm of chromosome 19. There are few portion differs from the result of cytogenetic clinical resemblances between our elderly analysis in cultured cells which is based on a patient with ring 19 mosaicism and the small sample of metaphases obtained from non-mosaic child reported by Quack et all'7; PHA stimulated T lymphocytes, and which neither patient has any major congenital showed not more than 28% abnormal cells. We malformation, both have moderate mental currently use CGH to determine the origin of handicap, but the striking difference is that our cytogenetically unidentifiable chromosomes elderly patient has reduced stature and propor- and so far have obtained successful results in tionately reduced head circumference, whereas 10 cases with different sizes and percentages of the subject of the report of Quack et all'7 has ring and marker chromosomes (unpublished length, weight, and head circumference more data). However, since in CGH the centromeric than 3 SD above the mean. Quack et all'7 regions are suppressed, the method would not estimated that the IQ of their proband was 69 be applicable to ring or marker chromosomes and reported particular difficulties with lan- composed almost entirely of centromeric guage, which is reminiscent of the findings in heterochromatin. our patient whose psychological evaluations The classical explanation for formation of a showed no evidence of a behavioural pheno- centric ring involves breaks in both arms of the type and only some evidence of age related chromosome with subsequent fusion of the gradual decline in intellect. In summary, in proximal ends to form a ring and loss of the three cases non-specific moderate to mild material distal to the breakpoints. More mental retardation or developmental delay has recently the possibility of U type fusions or a U been caused by mosaicism for an abnormal, with transverse supernumerary chromosome 19. type fusion in combination So far the approach to determining the misdivision of the centromere has been put origin of cytogenetically unidentifiable chro- forward as a mechanism for the generation of mosomes has involved stepwise FISH using small ring chromosomes.3" In the case de- forward painting methods with alpha satellite, scribed here, no short arm material could be single copy, or whole chromosome painting seen in the ring chromosome either by CGH or probes, or reverse chromosome painting proce- by FISH using short arm paint, suggesting a dures with either flow sorting or microdissec- breakpoint at or immediately adjacent to the http://jmg.bmj.com/ tion of the particular chromosome.'" Micro- centromere. No more detailed studies have yet dissection followed by DOP-PCR been undertaken to define this breakpoint amplification of the dissected DNA is increas- more precisely. CGH studies indicate that vir- ingly being used and its modified approaches tually the entire long arm of chromosome 19 is are reported to be so efficient that just one dis- present in the ring, although by FISH the telo- sected DNA fragment is sufficient for a meric region is shown not to be present. We successful analysis.9 However, not all diagnos- have previously shown that partial trisomy or on September 26, 2021 by guest. Protected copyright. tic cytogenetics laboratories have easy access to partial monosomy of as little as 4-5 Mb can be microdissection technology. Here we present detected in telomeric regions by CGH.'6 The an alternative approach which is not dependent fact that CGH has not shown any variation in on patient metaphase and is a genome wide copy number of any part of 19q suggests that screening technique enabling the result to be the breakpoint is within 5 Mb of the telomere, achieved in just one experiment. Many image and that the loss of long arm material in the analysis systems now in use in cytogenetic formation of the ring chromosome is beyond laboratories include software for CGH, and the resolution of CGH. However, although we results of analysis are available in three working have precisely identified the identity of the ring days, which is comparable to the time required chromosome, we are not able to distinguish the for microdissection. Where further confirma- mechanism by which it arose. tion by FISH is necessary one more day is The first series of FISH experiments using required. empirically selected centromeric probes in- An advantage of CGH over microdissection cluding one for chromosome 19 failed to indi- is that, as shown here, it can provide an cate the origin ofmicrodissection. This may be estimate of the proportion of cells containing explained in part by the low percentage of cells the ring chromosome in the tissue studied. containing the ring and difficulties in identify- This information is laborious to obtain by ing it with certainty in some mitoses. Also the chromosome analysis or interphase FISH. The alpha satellite probe hybridising to chromo- minimum percentage of abnormal cells that somes 1, 5, and 19 was used in the study, since can be detected by CGH has not yet been at that time it was the only available defined, but based on this report and our centromeric probe for chromosome 19. It is unpublished data, as little as 15% mosaicism in possible that a small change in stringency con- cultured metaphase cells could be detected. ditions caused some loss of signal for chromo- Theoretically, using a medium restrictive fluo- some 19. 840 Ghaffari, Boyd, Connor, et al

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