Multiple forms of atypical rearrangements generating supernumerary derivative chromosome 15
© Wang et al; licensee BioMed Central Ltd. 2008
Received: 27 August 2007
Accepted: 04 January 2008
Published: 04 January 2008
Maternally-derived duplications that include the imprinted region on the proximal long arm of chromosome 15 underlie a complex neurobehavioral disorder characterized by cognitive impairment, seizures and a substantial risk for autism spectrum disorders. The duplications most often take the form of a supernumerary pseudodicentric derivative chromosome 15 [der(15)] that has been called inverted duplication 15 or isodicentric 15 [idic(15)], although interstitial rearrangements also occur. Similar to the deletions found in most cases of Angelman and Prader Willi syndrome, the duplications appear to be mediated by unequal homologous recombination involving low copy repeats (LCR) that are found clustered in the region. Five recurrent breakpoints have been described in most cases of segmental aneuploidy of chromosome 15q11-q13 and previous studies have shown that most idic(15) chromosomes arise through BP3:BP3 or BP4:BP5 recombination events.
Here we describe four duplication chromosomes that show evidence of atypical recombination events that involve regions outside the common breakpoints. Additionally, in one patient with a mosaic complex der(15), we examined homologous pairing of chromosome 15q11-q13 alleles by FISH in a region of frontal cortex, which identified mosaicism in this tissue and also demonstrated pairing of the signals from the der(15) and the normal homologues.
Involvement of atypical BP in the generation of idic(15) chromosomes can lead to considerable structural heterogeneity.
The segmental nature of the rearrangements arises due to a series of large transcribed repeats derived from the HERC2 locus as well as low copy repeats of chromosome 15 (LCR15s), which provide the basis for the stereotypic nature of most rearrangements involving this region [8–10]. It is believed that the LCR cause misalignment during meiosis I, which leads to unequal but homologous recombination events involving both sister chromatid or interchromosomal exchanges. In addition to the repeat-mediated illegitimate recombination events, a region with a high rate of recombination has been identified within the PWS/AS critical region. This region lies near the D15S122 and the GABRB3 loci, which lie ~1 Mb apart, yet show genetic distances of ~4 cM in females and ~1 cM in males, suggesting a recombination hotspot in females .
We recently developed an array comparative genomic hybridization (array-CGH) tool to examine duplications of chromosome 15q . Combining this array with standard molecular and cytogenetic strategies, we identified four patients with atypical forms of idic15 chromosomes that lead to varying degrees of segmental aneuploidy for the proximal long arm and that indicate that additional crossovers may occur within the idic(15) chromosome.
Clinical Characteristics of subjects with atypical duplications
Chronological Age at examination
Mean Mental Age
Epicanthus, low nasal bridge, unfolded ears
Infantile spasms, Lennox Gastaut
Epicanthus, unilateral cryptorchidism
Epicanthus, low nasal bridge, unfolded ears
A fixed surgical sample of frontal cortex was available for this patient and was used for interphase FISH with probes CEP15 and GABRB3 (Figure 4G). These studies confirmed mosaicism in brain with 40.3% of cells showing less than or equal to two spots for the CEP15 and GABRB3 probes. Pairing of the signals was also examined. Pairing of the GABRB3 loci near the nucleolus was evident in both euploid and aneuploid neuronal nuclei, although some extra GABRB3 signals remained unpaired in der(15) containing nuclei.
The cases described here indicate a wide range of complexity of duplication chromosomes derived from chromosome 15. In patient 00.16, the two der(15) were dicentric and each appeared to carry one extra copy of the region between BP2-BP3. This was further supported by the dosage measurements derived array CGH, which were consistent with overall tetrasomy of the duplicated region. However using array CGH without cytogenetic analyses would have missed the structural anomalies for the der(15)s. The molecular mechanisms that generated these idic(15) chromosomes are speculated to involve an interchromosomal exchange during meiosis, with a subsequent segregation error early in development, leading to two apparently identical idic(15) chromosomes.
Cases 99.30 and 03.46 have internal rearrangements within the der(15) that led to partial hexasomy for the involved segments. For 99.30, the increase in dosage for the region within the BP3-BP4 region was clearly detected by the array CGH studies and subsequently confirmed using FISH. Notably, at diagnosis, the complexity of the der(15) was not identified because the FISH probe used detected only the proximal BP2-BP3 region that was not included in the hexasomic segment. Phenotypically, this child performs on the lower end of the cognitive spectrum by comparison with other cases of idic(15) and is microcephalic (OFC < 2nd centile for age), which is not typical of the syndrome. The der(15) carried by 03.46 is distinctive in that it appears to include 4 tandemly arranged segments, each of which includes the region proximal to BP1. While this region is typically included twice in the region between the centromere and BP1 in a typical idic(15) chromosome, it is unexpected that it would be included in the internally rearranged segments, indicating that the mechanisms that gave rise to this der(15) involved unique recombination events that lie outside the LCR. Pairing of the der(15) chromosomes in neurons has not previously been described. The proximity of the duplication chromosomes with the normal homologues is consistent with maintenance of perinucleolar organization of 15q11-13, despite the lack of the ribosomal RNA gene clusters in the der(15) chromosome. Homologous pairing of 15q11-13 is predicted to be important for the maintenance of biallelic expression of GABAA receptor genes (Hogart et al, 2007), but the effect of der(15) chromosomes on GABAA receptor gene expression is currently unknown. Previous expression studies in lymphocytes that demonstrated maternal expression of the UBE3A gene on idic(15) chromosomes.
For the family of 99.10, the large r(15) that was identified in the mother likely contributed to the subsequent abnormal crossover during oogenesis that led to the formation of the idic(15) in her daughter. The mother in this family is phenotypically normal, which likely reflects her low level mosaicism and may also represent a paternal origin of the r(15). Because grandparental samples were not available, it is not possible to determine the origin of the r(15). Array CGH performed with the prior knowledge of the r(15) was able to detect the presence of the r(15) however the low level dosage increase would not be expected to be detected clinically.
In two probands, 00.16 and 99.30, a shift in biallelic and monoallelic maternal contributions were observed, with change occurring in the BP2-BP3 interval in a region that has previously been identified as a recombination hotspot on chromosome 15q11-q12 in females. For 00.16, this likely represents an recombination event on the normal maternal homologue, although this cannot be definitively confirmed based on morphology of the der(15) chomosomes. For case 99.30, this shift between monoallelic and biallelic maternal contribution occurs in the region where the internal rearrangement of the der(15) is detected, hence increasing the likelihood a complex interchromosome and intrachromosomal exchange was involved in the generation of the the idic(15) chromosome. In summary, the cases described here indicate that not all der(15) chromosomes arise through nonhomologous allelic recombinations mediated by LCR present within the chromosome 15q11-q13 region.
Subjects and cell lines
The subjects were recruited to the study after clinical diagnosis of duplications of 15q11-q13 by karyotype combined with fluorescence in situ hybridization (FISH) analysis. Peripheral blood samples were collected from the patients and their parents following informed consent using protocols approved by the Institutional Review Board at the University of California, Los Angeles and the Alfred I. duPont Hospital for Children. Lymphoblast cell lines were established by transformation with Epstein-Barr virus using standard techniques. For one patient (03.46), a paraffin embedded surgical sample of frontal cortex was available. Phenotyping was performed in the child's home using the Mullen Scales of Early learning to examine cognition, and the autism diagnostic interview-revised  and autism diagnositic observation scale-generic for the diagnosis of autism. Additional clinical data were obtained from physical examination and medical record review.
STS markers used for genotyping
D15S541, D15S542, D15S1035, D15S17
D15S11CA, D15S646, D15S817, D15S1021, D15S122, D15S210, D15S986, D15S1513, GABRB3, D15S97, GABRA5, D15S822, D15S975, D15S219, D15S156
D15S1019, D15S815, D15S1048, D15S1043
D15S165, D15S184, D15S1013, D15S1031
Distal to BP5
D15S144, D15S1007, D15S123
The imprinting status of the extra genomic material was ascertained using Southern blot analysis with the SNRPN probe. Genomic DNA from blood was isolated using a Puregene DNA isolation Kit (Gentra Systems, Minneapolis, Minnesota) and resuspended in TE pH 8.0. DNA (10 μg) was digested with XbaI and NotI, a methylation sensitive restriction enzyme. Digestions were resolved on a 0.8% TAE gel and then transferred onto Hybond N+ nylon membranes (Amersham Pharmacia, Piscataway, NJ). Following transfer, DNA was UV crosslinked onto the membrane. Probes were labeled by random oligonucleotide priming using the Prime-It Random Labeling Kit (Stratagene, La Jolla, CA) and α-32P-dCTP (Perkin-Elmer. Waltham, MA) and hybridized onto the blots using PerfectHyb Plus Hybridization Buffer (Sigma Aldrich, St. Louis, MO). Hybridized blots were exposed onto phosphorimager screens (Amersham Pharmacia) overnight and images were scanned on a Storm phosphorimager (Amersham Pharmacia). Analysis was performed using the ImageQuant software package (Amersham Pharmacia). Presence of the maternal (4.2 kb) and paternal band (900 bp) was used to exclude PWS and AS. After measuring the signal intensities of both the maternal and paternal band, a maternal/paternal ratio was derived to determine dosage of the SNRPN region and the methylation status of the duplicated genomic region.
Fluorescence in situ hybrization (FISH)
FISH analysis was performed as previously described. Metaphase spreads or interphase nuclei were prepared from lymphoblastoid cell lines or phytohemaglutinin (PHA) stimulated cultures of peripheral leukocytes. DNA from BAC or cosmid clones (Figure 1) from chromosome 15 was nick translated and co-hybridized with a centromere probe for chromosome 15 (pcm15) generously provided by Dr. Mariano Rocchi (University of Bare, Italy). Signals on the normal homologues and the der(15) chromosomes gave were scored visually. Hybridization was detected by epifluorescence using a Leica DM RXA2 microscope using OpenLab 3.1.3 software (Improvision, Lexington, MA) or (patient 00-16) using a Leica DMX microscope equipped with a CCD camera and IPLab software (Scanalytics, Vienna, VA). A minimum of 20 metaphase spreads was examined for each probe. In mosaic samples, at least 100 nuclei were counted.
Brain tissue FISH
Paraffin-embedded fixed frontal cortex from case 03.46 (ID: SS-99-5542) was sectioned at 5 μm onto glass slides. Slides were baked overnight at 56°C, then placed in four 5 minute washes with xylene, then two 5 minute washes with 100% ethanol, then 1 hour in 95°C in antigen retrieval solution (DAKO). Slides were then post-fixed in Histochoice for 90 minutes, then washed 5 minutes in 1× phosphate buffered saline (PBS). Slides were dehydrated in 50%, 70%, 90%, 100% ethanol (10 minutes each), then dried at 50°C. A probe mixture containing 1 μl each probe (CEP15 and GABRB3, Vysis, Inc.), 2 μl ddH2O, 7 μl LSI/WCP buffer (Vysis, Inc.) was warmed to 37°C, then added to the slide, coverslipped, and sealed with rubber cement. Probe and cells were simultaneously denatured at 85°C for 2 minutes on a slide cycler (Hybaid). Slides were incubated overnight at 37°C, the washed in 50% Formamide/50% 2× SSC thrice for 5 minutes, 0.5× SSC for 5 minutes, and 0.5× SSC/0.1% IGEPAL for 5 minutes, all at 46°C, pH 7.6. 250 μg/ml RNAse was added to the slides, coverslipped and incubated at 37°C for 30 minutes, then 5 minutes in 1× PBS and air dried. Slides were mounted with 5 μg/ml DAPI in Vectashield (Vector Laboratories), coverslipped and sealed with nail polish. Slides were analyzed on an Axioplan 2 fluorescence microscope (Carl Zeiss, Inc, NY) equipped with a Sensys CCD camera (Photometrics, Tucson, AZ), appropriate fluorescent filter sets, and automated xyz stage controls. The microscope and peripherals were controlled by a Macintosh running IPLab Spectrum (Scanalytics, Vienna, VA) software with Multiprobe, Zeissmover, and 3D extensions. Images were captured for blue, green, and red filters at one edge of the specimen, then repeated at 0.4 micron sections through the depth of the tissue. Each image stack was digitally deconvolved to remove out-of-focus light using HazeBuster software (Vaytek, Fairfield, IA). Following haze removal, image stacks for each fluorophore were merged and stacked to create a two-dimensional image representing all of fluorescence within the section.
Array Comparative Genomic Hybridization (Array-CGH)
Array CGH was performed for each patient using custom BAC arrays as described . The log2Test/Reference (log2T/R) ratios were calculated for each array probe and plotted linearly. Dosage was determined using the standard curve previously reported . Positions of the array probes were based on the April 2003 genome assembly.
List of Abbreviations
Bacterial Artifical Chromosome
Comparative Genomic Hybridization
derivative chromosome 15
Fluorescence in situ hybridization
gamma butyric acid
- Int dup(15):
interstitial duplication (15)
Low copy repeats
phosphate buffered saline
Prader Willi syndrome
ring chromosome 15
We are grateful to the families who participated in these studies and the IDEAS support group. We thank Suzanne M. Mann, PhD for her assistance with the FISH studies for patient 00-16. The authors are supported by U19-HD35470 (NW, JD, MS and NCS), U19-RR (NCS) and Nemours (BM, JD).
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