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Editor—Application of chromosome painting has enabled confirmation that an additional segment in a presumed tandem duplication originates from the rearranged chromosome. More recent studies to determine more accurately the exact amount of duplication with cosmid FISH probes have, in a few instances, shown that some of the presumed duplications were in fact triplications of smaller segments.1-7 In some of these patients, unequal distances between the FISH signals showed that the middle segment of the tandemly arrayed three segments was in the opposite orientation to the two flanking segments,1 3 4 thus providing clues to the possible mechanism of formation.
Here we report on the clinical, cytogenetic, and molecular analysis of a patient displaying the same type of triplication for segment 13q14 including the retinoblastoma gene, again with opposite orientation of the middle segment.
The proband was born at term after an uneventful pregnancy to healthy, consanguineous, Turkish parents (third cousins). At the birth, the mother was 23 and the father was 28 years old. The patient's birth weight was 2700 g (<10th centile). At the age of 4 years, she was referred to a paediatric hospital for evaluation of developmental delay. Her weight (8 kg), height (85.0 cm), and head circumference (43.5 cm) were all far below the 3rd centile. Her cognitive abilities were estimated to be at the level of a 2 year old (IQ of about 50). Clinical assessment showed the following findings (fig 1): low frontal hairline, prominent antihelices and hypoplastic lobules of the ears, upward slanting palpebral fissures, deep set eyes, a capillary haemangioma next to the right eyebrow, a bulbous nasal tip, thin upper lip, pointed chin, excessive dental caries, right transverse palmar crease, diminished flexion in the metacarpophalangeal joints of both thumbs, and hypertrichosis of the legs.
Chromosome analysis was performed on GTG banded metaphase preparations of blood lymphocyte cultures of the patient and her parents by standard techniques.
FISH was carried out using probes from four loci on chromosome 13 according to standard protocols (Oncor® Inc) or as previously described.8 9 Loci and their corresponding cosmid probes included D13S118 (c118), the retinoblastoma geneRB1, D13S319 (c319), and D13S25 (c25), all mapping to 13q14. Cosmid c118 maps to 13q14.1-q14.2 proximal toRB1, while cosmids c319 and c25 both map to 13q14.3 about 1 Mb distal to the RB1locus.10
FISH analysis was performed using a Zeiss Axioplan epifluorescence microscope and images were recorded either by conventional microphotography or by Photometrics CCD KAF camera (Tuscon, AZ, USA), controlled with Smart Capture imaging software (Vysis Inc, Downers Grove, IL, USA).
Genomic DNA was extracted from peripheral blood of the proband and her parents by standard methods. PCR analysis was performed using primers which amplify dinucleotide repeat polymorphisms. The following loci which map between bands 13q12 and 13q32-q34 were tested: D13S221, D13S218, D13S118, D13263, D13S155, D13S284, D13S153, D13S137, D13S124, D13S162, D13S170, and D13S173. PCR products were separated on a 6% polyacrylamide/urea gel and visualised by silver staining.
GTG banding analysis of the patient's metaphase spreads (resolution of about 400-500 bands/haploid karyotype) showed a female karyotype with 46 chromosomes in all 30 cells examined. One homologue 13 showed an approximate 1/4-1/3 increase in length owing to an additional segment in the middle of the long arm. Two narrow bands divided the additional segment into three equal parts, suggesting a triplication of band 13q14 (fig 2). The karyotypes of both parents were normal.
Hybridisation with the RB1 gene probe showed one signal on the normal chromosome and three signals and thus three RB1 copies on the rearranged homologue. The distances between the three signals appeared to be unequal; the distance between the first and second signals seemed to be shorter than that between the second and third signals. Cohybridisation of the RB1 gene probe with differently labelled cosmids c25 or c319, as well as cohybridisation of cosmids c118 and c319, confirmed an interstitial triplication of the whole of band 13q14 (fig 2). In addition, dual colour FISH clearly showed that the middle segment was inverted in orientation; the order of signals was RG-GR-RG or GR-RG-GR (R stands for red; G stands for green). An order of RG-RG-RG or GR-GR-GR would be expected if the triplication had occurred in direct orientation. Unequal distance between three pairs of signals, with the first two in a row being very close, but both being quite far apart from the third pair of signals, indicates a distal location of the RB1 gene within band 13q14 (fig 2). FISH and DAPI banding (=850 bands/karyotype) placed theRB1 locus at 13q14.2. Combining the pattern of GTG bands with the results of DAPI banding, the proximal breakpoint appears to be very distally located in 13q13.3, while the distal breakpoint appears to be very proximally located in 13q21.1 (fig2).
The patient's karyotype can be described as: 46,XX,?trp(13)(q?14q?14)de novo.fish trp(13)(pter→ q21.1::q21.1→q13.3::q13.3→qter)(D13S118+++,RB1+++, D13S319+++, D13S25+++).
The results of microsatellite polymorphism analysis are shown in table1. Two of the 12 loci showed inheritance of one maternal and two paternal alleles. Locus D13S284, which maps to 13q14.3, displayed three distinct alleles, two being paternal in origin (fig 3). At locus D13S155, which maps to 13q14.3-q21.2, the paternal band was clearly stronger than the maternal band. At locus D13S263, which maps to 13q14.1-q14.2, the paternal band was possibly stronger than the maternal one (data not shown). Normal biparental inheritance of loci proximal (D13S221) and distal (D13S170, D13S173) to 13q14 was seen. These results indicate that the triplication of 13q14 was paternal in origin and that both paternal chromosomes 13 were involved in its formation.
Clinical observations suggest that, in general, tetrasomy of an autosomal segment causes a similar, but more severe clinical picture than trisomy of the same segment. This has been shown, for example, for 9p, 18p, and 15(q12-q13).1 11 Further, for some segments (8p, 12p) tetrasomy is viable only in a mosaic state while trisomy without mosaicism is compatible with survival. No other patients with tetrasomy for segment 13q14 are yet known. Trisomy of this segment was only detected following cytogenetic investigation of families with several members affected with retinoblastoma; unbalanced familial insertional translocations resulting in 13q14 deletion caused retinoblastoma while those resulting in 13q14 duplication caused a very mild clinical phenotype or even no anomalies.12-14 The patients were described as either having “no definite clinical syndrome”,12 being normal,13 or normal with short stature.14 Thus, the mild but still distinct phenotype of our proband with triplication as compared to the above mentioned cases with duplication of 13q14 is in accordance with the previous observations with other chromosomal segments. Duplication of a larger segment (13q12-q22) including 13q14 was also found in family investigations because of familial occurrence of retinoblastoma.15 Not unexpectedly because of the larger size of the duplication, these patients showed mild dysmorphism and mild to moderate mental retardation. Patients with a proximal duplication, dup(13)(pter-q14/q21), show a non-specific pattern of mostly minor anomalies and moderate mental retardation, while patients with a distal 13q duplication including 13q14, for example, dup(13)(q14→qter), show multiple congenital anomalies including postaxial polydactyly and profound mental retardation.16
Tetrasomy of (13)(q14.3→q22), that is, most of segment 13q21, in combination with duplications of the adjacent proximal (13q14.1-q14.3) and distal (13q22-qter) segments was determined in a newborn girl with a complex de novo rearrangement.17 The proband died shortly after birth with multiple major malformations including a malformation of the brain stem, cerebellar hypoplasia, cloudy corneae, aniridia, microphthalmia and other ocular anomalies, intestinal malrotation, cystic horseshoe kidneys, patent urachus, polydactyly of the fingers and toes, and many others. In this case, it is impossible to attribute single anomalies to duplication versus triplication of specific 13q segments present in a hyperdiploid state.
While direct or inverted tandem duplications could be formed through unequal crossover or unequal sister chromatic exchange,18 triplications of this kind require a more complex mechanism of origin. For a case of triplication of 15q11-q13, meiotic recombination between an inv dup(15)(q13) chromosome and a normal 15 with subsequent loss of the marker was proposed.1 A similar mechanism has also been proposed to explain amplification of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells.19 Another possible mechanism would be that the parent from whom the aberration stems (in our case the father) would either carry a constitutional paracentric inversion of the triplicated segment or such an inversion would be formed in a prezygotic cell. Although banded chromosome examinations of the father of our proband yielded normal results, a paracentric inversion of just 13q14 could not be excluded unless bicolour FISH studies were performed, which in this case was not possible. Two crossovers within the inversion loop with a U type exchange would result in a tandem triplication with an inverted middle segment.
This type of triplication has so far been reported for the following chromosomal segments: 2q11.2-q21,7 2q37,25p14-p15.33,3 7p21.2-p21.3,49p13-p22,5 10q26,6 13q14 (present report), and 15q11-q13.1 20 21 It is interesting to note that in several cases an unequal distance between two FISH signals (the proximal and the middle versus the middle and the distal) indicated that either the middle segment was inverted or, less likely, the middle segment was in normal orientation, but the proximal and distal segments were inverted,1 4 6 probably, according to the illustrations, the cases of Rauch et al 2 and Harrison et al 3 and the present report. In the other patients, no special attention was paid to the issue of direct versus inverted triplication. In the case of direct orientation of all three segments, the distances between the signals should be of equal length; however, even in the case of an inverted middle segment, the distances would only be of equal length if the FISH probes map to the middle of the triplicated segment at equal distances from the two ends.
It is likely that a proportion of cases of segmental triplication are incorrectly interpreted as duplication while in fact there is triplication of a smaller segment. This could also account for part of the phenotypic variability observed between such patients with presumed similar phenotypes, especially with respect to interstitial segments for which there are few familial insertional translocations and thus proven instances of duplication. Banded chromosome analysis alone will in very few cases allow discrimination between duplication and triplication. We therefore propose that FISH studies using probes mapping to the presumed duplicated segment should constitute part of the diagnostic work up following determination of a tandem duplication through banded chromosome examination and chromosome painting with whole chromosome probes.
The authors thank Drs P Lichter and S Stilgenbauer for YACs from the 13q14 segment. The investigation was supported by the Swiss National Foundation, grant Nos 32-45604.95 and 32-37798.93.