Ring chromosome 15: characterization by array CGH

Ring chromosome 15 [r(15)] is an uncommon finding with less than 50 patients reported. Precise genotype–phenotype correlations are problematic because of the difficulties in determining the extent of euchromatic loss, the level of mosaicism, and the influence of the timing of ascertainment. We report two discordant examples of r(15) patients. In the first case, prenatal diagnosis of a de novo r(15) was made during the second trimester: mos 46,XX,r(15)(p11.2q26)[32]/45,XX,-15[13]/47,XX,r(15)(p11.2q26)x2[3]/46,XX,dic r(15)(p11.2q26p11.2q26[1]/46,XX[2]. Postnatal follow-up revealed extremely small stature, heart defects, and developmental delay. Patient 2 was a 31-year-old short-statured female who was living independently: 46,XX,r(15)(p11q26). Both cases showed loss of the 15q subtelomeric region by fluorescence in situ hybridization (FISH). To investigate the discordance in phenotypes between the two patients, we undertook array comparative genomic hybridization (array CGH) analyses to more fully characterize the deletions associated with these otherwise structurally indistinguishable r(15) chromosomes from conventional cytogenetic analyses and fluorescence in situ hybridization (FISH) studies. By array CGH, patient 1 showed deletion of multiple contiguous clones predicting an approximately 6 Mb deletion of distal 15q. In contrast, patient 2 showed loss of just the 15q subtelomeric clone and an interstitial clone by array CGH confirming that the severity of the phenotype correlated with the size of the deletion at the molecular level. These cases illustrate the utility of array CGH characterization for determining the size of the associated deletion in ring chromosomes and for facilitating phenotype–genotype correlations.     

Autistic disorder and chromosome 15q11-q13: construction and analysis of a BAC/PAC contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2-10/10,000 individuals. Chromosome 15q11-q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the gamma-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11-q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11-q13.     

The structure of duplications on human acrocentric chromosome short arms derived by the analysis of 15p

We report the molecular analysis of a 130-kb DNA region containing a junction between beta and non-beta satellite DNA from chromosome 15p. The genomic region is characterized by beta satellite blocks intermingled with variants of the D4Z4 repeat, and duplicons from 4q24 and 4q35. Besides the p-arm of acrocentric chromosomes, the duplicons showed a wide genomespread involving pericentromeric, sub-telomeric, and interstitial regions. In this regard, the paralogous sequences were characterized by a high similarity index (96%), thus indicating a recent transposition during the evolution. The acrocentrics differedwith regard to the location of the 4q24 paralogous region, since it mapped on the p-arm of chromosomes 13-15 and 21, but only on 22q11.2. Conversely, the 4q35 duplication marked the p-arm of all the acrocentrics. In different individuals, the short arm of acrocentric chromosomes revealed a great variability of sequence representation and location at p11 and/or p13 for both the 4q24 and 4q35 duplications. The studied genomic region from chromosome 15p, of which a contig of approximately 200 kb has been derived, could lead to more detailed investigations into the sequence organization and possible biological function of chromosome regions that are located close to the rDNA array.     

Isolation and molecular analysis of inv dup(15) and construction of a physical map of a common breakpoint in order to elucidate their mechanism of formation

An inverted duplication of chromosome 15 [inv dup(15)] is the most common supernumerary marker chromosome, comprising approximately 50% of all chromosomes in this class. Structurally, the inv dup(15) is a mirror image with the central axis defining a distal break within either the heterochromatic alpha-satellite array or along the euchromatin in the long (q) arm of the chromosome. There are several types of inv dup(15), classified by the amount of euchromatic material present. Generally, they are bisatellited, pseudodicentric and have a breakpoint in 15q11-q14. A suggested mechanism of formation of inv dup(15) involves illegitimate recombination between homologous chromosomes followed by nondisjunction and centromere inactivation. The proximal portion of chromosome 15 contains several low-copy repeat sequence families and it has been hypothesized that errors in pairing among these repeats may result in structural rearrangements of this chromosome including the inv dup(15). To test this hypothesis and to determine the mechanism of formation, the inv dup(15) from four cases was isolated in somatic cell hybrids and polymerase chain reaction microsatellite markers were used to determine the origin of exchange. Two appeared to result from interchromosomal and two from intrachromosomal exchange, one of which occurred post-recombination. In addition, a detailed physical map of the breakpoint region in the largest inv dup(15) was constructed placing eight new sequence-tagged sites and ten new bacterial artificial chromosome markers in the region.     

A Physical Map of 15 Loci on Human Chromosome 5q23-q33 by Two-Color Fluorescence in Situ Hybridization

The q23-q33 region of human chromosome 5 encodes a large number of growth factors, growth factor receptors, and hormone/neurotransmitter receptors. This is also the general region into which several disease genes have been mapped, including diastrophic dysplasia, Treacher Collins syndrome, hereditary startle disease, the myeloid disorders that are associated with the 5q-syndrome, autosomal-dominant forms of hereditary deafness, and limb girdle muscular dystrophy. We have developed a framework physical map of this region using cosmid clones isolated from the Los Alamos arrayed chromosome 5-specific library. Entry points into this library included 14 probes to genes within this interval and one anonymous polymorphic marker locus. A physical map has been constructed using fluorescence in situ hybridization of these cosmids on metaphase and interphase chromosomes, and this is in good agreement with the radiation hybrid map of the region. The derived order of loci across the region is cen-IL4-IL5-IRF1-IL3-IL9-EGR1-CD14-FGFA-GRL-D5S207-ADRB2-SPARC-RPS14-CSF1R-ADRA1, and the total distance spanned by these loci is approximately 15 Mb. The framework map, genomic clones, and contig expansion within 5q23-q33 should provide valuable resources for the eventual isolation of the clinically relevant loci that reside in this region.     

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): high-resolution physical and transcript map of the candidate region in chromosome region 13q11

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is a neurodegenerative disease frequent in northeastern Québec. In a previous study, we localized the disease gene to chromosome region 13q11 by identifying excess sharing of a marker allele in patients followed by linkage analysis and haplotyping. To create a detailed physical map of this region, we screened CEPH mega-YACs with 41 chromosome 13 sequence-tagged-sites (STSs) known to map to 13q11-q12. The YAC contig, composed of 27 clones, extends on the genetic map from D13S175 to D13S221, an estimated distance of at least 19.3 cM. A high-resolution BAC and PAC map that includes the ARSACS critical region flanked by D13S1275 and D13S292 was constructed. These YAC and BAC/PAC maps allowed the accurate placement of 29 genes and ESTs previously mapped to the proximal region of chromosome 13q. We confirmed the position of two candidate genes within the critical region and mapped the other 27 genes and ESTs to nearby intervals. Six BAC/PAC clones form a contig between D13S232 and D13S787 for sequencing within the ARSACS critical region.     

Arginase deficiency manifesting delayed clinical sequelae and induction of a kidney arginase isozyme

De novo chromosome structural abnormalities cannot always be diagnosed by the use of standard cytogenetic techniques. We applied a previously developed chromosome-band-specific painting method to the diagnosis of such rearrangements. The diagnostic procedures consisted of microdissection of an aberrant chromosomal region of a given patient, polymerase chain reaction (PCR) amplification of the dissected chromosomal DNA, and subsequent competitive fluorescence in situ hybridization (FISH) using the PCR products as a probe pool on metaphase chromosomes from the patient and/or a karyotypically normal person. With this strategy, we studied 6 de novo rearrangements (6p+, 6q+, 9p+, 17p+, +mar, and +mar) in 6 patients. These rearrangements had been seen by conventional banding but their origin could not be identified. In all 6 patients, we successfully ascertained the origin. Using an aberrant region-specific probe pool, FISH signals appeared on both the aberrant region and a region of another specific chromosome pair. A reverse probe pool that was generated through the microdissection of normal chromosomes at a candidate region for the origin of the aberration hybridized with both the aberrant and the candidate regions. We thus diagnosed one patient with 17p+ as having trisomy for 14q32-qter, one with 9p+ as having trisomy for 12pter-p12, one with 6q+ as having a tandem duplication (trisomy) of a 6q23-q25 segment, one with 6p+ as having a tandem duplication (trisomy) of a 6p23-q21.3 segment, one with a supernumerary metacentric marker chromosome as having tetrasomy for 18pter-cen, and the last with an additional small marker chromosome as having trisomy for 18p11.1 (or p11.2)-q11.2. The present targeted chromosome-band-painting method provides the simple and rapid preparation of a probe pool for region-specific FISH, and is useful for the diagnosis of chromosome abnormalities of unknown origin.     

From amplification to gene in thyroid cancer: a high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization.

Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. We now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. We used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3-6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKCepsilon), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKCepsilon as a previously unmapped candidate gene involved in thyroid tumorigenesis.     

Isolation of 1001 new markers from human chromosome 11, excluding the region of 11p13-p15.5, and their sublocalization by a new series of radiation-reduced somatic cell hybrids.

The determination of the physical map of human chromosome 11 will require more clones than are currently available. We have isolated an additional 1001 new markers in a bacteriophage vector from a somatic cell hybrid cell line that contains most of chromosome 11, except the middle of the short arm. These markers were localized to five different regions, 11p15-pter, 11p12-cen, 11q11-q14, 11q14-q23, and 11q23-qter, by a panel of previously characterized somatic cell hybrids. The region 11q11-14 harbors genes that have been shown to be important in breast cancer, B-cell lymphomas, centrocytic lymphomas, asthma, and multiple endocrine neoplasia, type 1 (MEN1). To determine the positions of the recombinant clones located there, we developed a new series of radiation-reduced somatic cell hybrids. These hybrids, together with those previously characterized, allowed us to map the 11q11-q14 markers into 11 separate segregation groups.     

Prader-Willi syndrome with an unusually large 15q deletion due to an unbalanced translocation t(4;15)

Prader-Willi syndrome (PWS) is a neurobehavioral disorder caused by deletions in the 15q11-q13 region, by maternal uniparental disomy of chromosome 15 or by imprinting defects. Structural rearrangements of chromosome 15 have been described in about 5% of the patients with typical or atypical PWS phenotype. An 8-year-old boy with a clinical diagnosis of PWS, severe neurodevelopmental delay, absence of speech and mental retardation was studied by cytogenetic and molecular techniques, and an unbalanced de novo karyotype 45,XY,der(4)t(4;15)(q35;q14),-15 was detected after GTG-banding. The patient was diagnosed by SNURF-SNRPN exon 1 methylation assay, and the extent of the deletions on chromosomes 4 and 15 was investigated by microsatellite analysis of markers located in 4qter and 15q13-q14 regions. The deletion of chromosome 4q was distal to D4S1652, and that of chromosome 15 was located between D15S1043 and D15S1010. Our patient's severely affected phenotype could be due to the extent of the deletion, larger than usually seen in PWS patients, although the unbalance of the derivative chromosome 4 cannot be ruled out as another possible cause. The breakpoint was located in the subtelomeric region, very close to the telomere, a region that has been described as having the lowest gene concentrations in the human genome.     

Duplication within chromosome region 15q11-q13 in a patient with similarities to Prader-Willi syndrome confirmed by region-specific and band-specific fish.

We report on a patient presenting with mental retardation and obesity and a proximal duplication of chromosome 15. The patient shared some clinical signs with Prader-Willi syndrome. With a region-specific paint, generated by microdissection, a duplication in region 15q11.2-q13 was shown to be present. Subsequently, FISH with probes localized to chromosome region 15q11.2-q12 and microsatellite analysis was used to characterize this chromosome aberration further and an insertion duplication within the region frequently deleted in Prader-Willi and Angelman syndrome was demonstrated.     

Molecular cytogenetic evidence for a common breakpoint in the largest inverted duplications of chromosome 15.

Chromosomes from 20 patients were used to delineate the breakpoints of inverted duplications of chromosome 15 (inv dup[15]) that include the Prader-Willi syndrome/Angelman syndrome (PWS/AS) chromosomal region (15q11-q13). YAC and cosmid clones from 15q11-q14 were used for FISH analysis, to detect the presence or absence of material on each inv dup(15). We describe two types of inv dup(15): those that break between D15S12 and D15S24, near the distal boundary of the PWS/AS chromosomal region, and those that share a breakpoint immediately proximal to D15S1010. Among the latter group, no breakpoint heterogeneity could be detected with the available probes, and one YAC (810f11) showed a reduced signal on each inv dup(15), compared with that on normal chromosomes 15. The lack of breakpoint heterogeneity may be the result of a U-type exchange involving particular sequences on either homologous chromosomes or sister chromatids. Parent-of-origin studies revealed that, in all the cases analyzed, the inv dup(15) was maternal in origin.     

Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages

Miscarriage is a condition that affects 10%-15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)-array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations.     

Deficiency,transposition, and duplication of one 15q region may be alternatively associated with Prader-Willi (or a similar) syndrome. Analysis of seven cases after varying ascertainment

Seven patients are described who have some or all of the symptoms of Prader-Willi syndrome. They were ascertained by varying criteria starting either from the clinical picture or from the identification of a chromosome abnormality involving the proximal portion of the long arm of chromosome 15. The chromosome abnormalities consisted of two balanced translocations (15;18 and 8;15), three unbalanced ones (15;18, 15;19, and 9;15), and one interstitial deletion of bands 15q11 and q12. The seventh case had an unidentified extra chromosome. These data and a review of the literature led to the conclusion that deficiency, transposition, and even duplication of the region(s) 15q11-q13 may all result in a syndrome which is identifiable with or similar to the Prader-Willi syndrome.     

A physical map of large segments of pig Chromosome 7q11–q14: comparative analysis with human Chromosome 6p21

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11-q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11-q14 region.     

Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations

Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.     

A novel combined 15q11.2 duplication and a bisatellited supernumerary marker derived from chromosome 22: Molecular characterization of the marker

Supernumerary marker chromosomes (SMC) are heterogeneous group of chromosomes which are reported in variable phenotypes. Approximately 70% originate from acrocentric chromosomes. Here we report a couple with recurrent miscarriages and a SMC originating from an acrocentric chromosome. The cytogenetic analysis of the husband revealed a karyotype of 47,XY+marker whereas the wife had a normal karyotype. Analysis of SMC with C-banding showed the presence of a big centromere in the center and silver staining showed prominent satellites on both sides of the marker. Apparently, microarray analysis revealed a 2.1 Mb duplication of 15q11.2 region but molecular cytogenetic analysis by fluorescence in situ hybridization (FISH) with whole chromosome paint (WCP) 15 showed that the SMC is not of chromosome 15 origin. Subsequently, FISH with centromere 22 identified the SMC to originate from chromosome 22 which was also confirmed by WCP 22. Additional dual FISH with centromere 22 and Acro-p-arm probes confirmed the centromere 22 and satellites on the SMC. Further fine mapping of the marker with Bacterial Artificial Chromosome (BAC) clones; two on chromosome 22 and four on chromosome 15 determined the marker to possess only centromere 22 sequences and that the duplication 15 exists directly on chromosome 15. In our study, we had identified and characterized a SMC showing inversion duplication 22(p11.1) combined with a direct tandem duplication of 15q11.2. The possible genotype–phenotype in relation with the two rearrangements is discussed.