Refined molecular characterization of the breakpoints in small inv dup(15) chromosomes

Inv dup(15) is the most common supernumerary marker chromosome in humans. To investigate the mechanism responsible for this frequent chromosome rearrangement, we characterized the breakpoints in 18 individuals with small inv dup(15) chromosomes [i.e., negative for the Prader-Willi (PWS)/Angelman syndrome (AS) critical region]. Since two proximal breakpoint regions (“hotspots”) for PWS/AS deletions have been previously identified with the most proximal 15q markers D15S541/S542 and S543, we hypothesized that formation of the small inv dup(15) chromosomes may involve one or both of these breakpoint hotspots. By analysis with S542, both breakpoint regions were found to be involved in approximately equal frequencies. In ten cases, the inv dup(15) was negative for S542 (Class I), indicating the breakpoint is between the centromere and the most proximal marker on chromosome 15. For the other eight cases, S542 was positive by fluorescence in situ hybridization (5/5) and/or microsatellite analysis (7/7), but S543 was negative (Class II). These two breakpoint regions appear to be the same as the two proximal breakpoints reported in the common PWS/AS deletions. To initiate cloning and sequencing of the Class II breakpoint, the gap in the yeast artificial chromosome (YAC) contig between S541/S542 and S543 was filled by screening the CEPH YAC and mega-YAC libraries. YACs 705C2 and 368H3 were found to bridge this gap, and therefore contain the more distal breakpoint region. The finding of consistent breakpoints in small inv dup(15), like that found in PWS/AS deletions, provides strong evidence for hotspots for chromosome breakage in this region. In addition, our results show that two extra copies (tetrasomy) of the region from 15cen to the euchromatic region containing S542 are present in individuals with Class II breakpoints. Since most individuals carrying a small inv dup(15) are phenotypically normal, the euchromatin region included in the small inv dup(15) chromosomes does not appear to contain genes with clinically significant dosage effects. Received: 23 May 1996 / Revised: 7 August 1996     

Preferential maternal derivation in inv dup(15)

Summary Eight patients are reported with a de nov extra inverted duplicated chromosome 15. The abnormal chromosome was considered to be the same in all cases, but its precise delineation remained uncertain and was defined as either 15qter15q12::15q1215pter or 15pter15q11::15q1315pter. Analysis with various techniques of the satellite regions of the bisatellited chromosomes demonstrated maternal derivation in six and paternal derivation in one of the seven families. A nonsister chromatid exchange between the two homologous chromosomes 15 is considered a likely origin of the inv dup(15) in the cases with maternal derivation; in the only case of paternal derivation, however, the abnormal chromosome originated from one single chromosome 15. The clinical findings confirm that patients with inv dup(15) have mental and developmental retardation and are frequently affected by seizures, while severe physical malformations are absent.     

Molecular cytogenetic analysis of inv dup(15) chromosomes, using probes specific for the Prader-Willi/Angelman syndrome region: clinical implications.

Twenty-seven cases of inverted duplications of chromosome 15 (inv dup [15]) were investigated by FISH with two DNA probes specific for the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region on proximal 15q. Sixteen of the marker chromosomes displayed two copies of each probe, while in the remaining 11 markers no hybridization was observed. A significant association was found between the presence of this region and an abnormal phenotype (P < .01). This is the largest study to date of inv dup(15) chromosomes, that uses molecular cytogenetic methods and is the first to report a significant association between the presence of a specific chromosomal region in such markers and an abnormal phenotype.     

Cytogenetic and clinical studies in five cases of inv dup(15)

Summary Inv dup(15) is a clinically significant bisatellited derivative of chromosome 15. Five unrelated patients with this abnormality are described and compared with ten confirmed and nine suspected cases in the literature. Mental and developmental retardation, hypotonia, behavioral disturbances, seizures, abnormal dermatoglyphics, and mild somatic anomalies were the most consistent findings. The extra chromosomes in our patients were identified with the aid of various techniques, including distamycin A/DAPI banding. A comparison of satellite polymorphisms suggested that the rearrangements frequently arose by meiotic nonsister chromatid exchange and second-division nondisjunction. A maternal origin was indicated in two cases, and parental ages were distinctly elevated.     

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.     

An analphoid marker chromosome inv dup(15)(q26.1qter), detected during prenatal diagnosis and characterized via chromosome microdissection

A small, mosaic, C-band negative marker chromosome was detected in amniocyte cultures during prenatal diagnosis due to advanced maternal age. Following spontaneous premature labor at 29 weeks gestation, a dysmorphic infant was delivered, with flat nasal bridge, short palpebral fissures, micrognathia, high forehead, low-set ears, telecanthus and corneal dystrophy. Additional folds of skin were present behind the neck, and feet, fingers and toes were abnormally long. The child died at age five days, after two days of renal failure. The origin of the marker chromosome was subsequently identified from a cord blood sample, via chromosome microdissection. Through reverse FISH, we found the marker to be an inverted duplication of the region 15q26.1-->qter. FISH with alphoid satellite probe was negative, while whole chromosome 15 paint was positive. Both ends of the marker chromosome were positive for the telomeric TTAGGG probe. These data, plus the G-banding pattern, identified the marker as an analphoid, inverted duplicated chromosome, lacking any conventional centromere. We discuss the etiology and clinical effects of this marker chromosome, comparing it to the few reported cases of "tetrasomy 15q" syndrome. We also discuss the possible mechanisms that are likely responsible for this neocentromere formation.     

A second case of inv(4)pat with both recombinants in the offspring: rec dup(4q) in a girl with Wolf-Hirschhorn syndrome and rec dup(4p)

In a girl presenting with features of Wolf-Hirschhorn syndrome, cytogenetic and molecular cytogenetic analysis revealed a rearranged chromosome 4 with monosomy of the distal bands 4pter-->4p16.2 and trisomy of the distal bands 4q35.1-->4qter [rec dup(4q)] due to a large, paternal pericentric inversion. In the following two pregnancies, prenatal diagnosis showed the same imbalance in one fetus and a reverse segmental imbalance [rec dup(4p)] in the other. We discuss the recombination risk of the given inversion with respect to the size of the inverted segment and the viability of the recombinants. The high frequency of recombinants in this family and others suggests a high recurrence risk in similar cases with large pericentric inversions comprising almost entire chromosomes.     

Supernumerary marker 15 chromosomes: a clinical,molecular and FISH approach to diagnosis and prognosis

Seventeen patients presenting with either de novo or familial supernumerary marker (mar) 15 chromosomes were shown by fluorescent in situ hybridization techniques (FISH) to have markers derived from and composed entirely of chromosome 15 material. Using a combination of conventional cytogenetics, FISH, Southern blotting and multiplex polymerase chain reaction (PCR) methods, it was possible to sub-classify the 17 mar(15)s into six distinct morphological and molecular groups. Analysis of DNA and metaphase spreads from the probands and their parents using probes and primers from the pericentromeric and Prader-Willi/Angelman syndromes critical regions (PWS/AS), clearly differentiated between marker 15 s which included the PWS/AS critical regions and those which did not. A direct correlation between the presence of the PWS/AS region in the mar(15) and severe mental retardation was observed. Based on these results, a system of classification of supernumerary marker 15 chromosomes is proposed.     

De novo unbalanced translocations in Prader-Willi and Angelman syndrome might be the reciprocal product of inv dup(15)s

The 15q11-q13 region is characterized by high instability, caused by the presence of several paralogous segmental duplications. Although most mechanisms dealing with cryptic deletions and amplifications have been at least partly characterized, little is known about the rare translocations involving this region. We characterized at the molecular level five unbalanced translocations, including a jumping one, having most of 15q transposed to the end of another chromosome, whereas the der(15)(pter->q11-q13) was missing. Imbalances were associated either with Prader-Willi or Angelman syndrome. Array-CGH demonstrated the absence of any copy number changes in the recipient chromosome in three cases, while one carried a cryptic terminal deletion and another a large terminal deletion, already diagnosed by classical cytogenetics. We cloned the breakpoint junctions in two cases, whereas cloning was impaired by complex regional genomic architecture and mosaicism in the others. Our results strongly indicate that some of our translocations originated through a prezygotic/postzygotic two-hit mechanism starting with the formation of an acentric 15qter->q1::q1->qter representing the reciprocal product of the inv dup(15) supernumerary marker chromosome. An embryo with such an acentric chromosome plus a normal chromosome 15 inherited from the other parent could survive only if partial trisomy 15 rescue would occur through elimination of part of the acentric chromosome, stabilization of the remaining portion with telomere capture, and formation of a derivative chromosome. All these events likely do not happen concurrently in a single cell but are rather the result of successive stabilization attempts occurring in different cells of which only the fittest will finally survive. Accordingly, jumping translocations might represent successful rescue attempts in different cells rather than transfer of the same 15q portion to different chromosomes. We also hypothesize that neocentromerization of the original acentric chromosome during early embryogenesis may be required to avoid its loss before cell survival is finally assured.     

Twenty-year cytogenetic and molecular follow-up of a patient with ring chromosome 15: a case report

ABSTRACT: INTRODUCTION: Ring chromosome 15 is a rare disorder, with only a few over 40 cases reported in the literature. There are only two previous reports of cases where patients with ring chromosome 15 have been followed-up. CASE PRESENTATION: We report here on the 20-year clinical and cytogenetic follow-up of a patient with a ring chromosome 15. Our patient, a Caucasoid Asian woman, presented with short stature, microcephaly, minor dysmorphic features, hyperextensible knees, generalized hirsutism, cafe-au-lait and small hypochromic spots spread over her face and the front of her chest and abdomen, dorsolumbar scoliosis and mild intellectual disability. She was followed-up from the age of eight to 28 years. When she was 27 years old, she was reported by her mother to present with compulsive overeating and an aggressive mood when challenged. Karyotyping revealed that the majority of her cells harbored one normal chromosome and one ring chromosome. Silver staining revealed the presence of the nucleolar organizer region in the ring chromosome. Ring loss and/or secondary aberrations exhibited a slight increase over time, from 4.67% in 1989 to 7.67% in 2009, with the presence of two monocentric rings, cells with interlocked rings, a dicentric ring, and broken or open rings. A genome-wide array technique detected a 5.5Mb deletion in 15q26.2. CONCLUSIONS: We observed that some phenotypic alterations in our patient can be associated with gene loss and haploinsufficiency. Other features may be related to different factors, including ring instability and epigenetic factors.     

Molecular cytogenetic characterization of the first reported case of an inv dup (4p)(p15.1-pter) with a concomitant 4q35.1-qter deletion and normal parents

Inverted duplications associated with terminal deletions are complex anomalies described in an increasing of chromosome ends. We report on the cytogenetic characterization of the first de novo inv dup del(4) with partial 4p duplication and 4q deletion in a girl with clinical signs consistent with “recombinant 4 syndrome”. This abnormality was suspected by banding, but high-resolution molecular cytogenetic investigations allowed us to define the breakpoints of the rearrangement. The terminal duplicated region extending from 4p15.1 to the telomere was estimated to be 29.27 Mb, while the size of the terminal deletion was 3.114 Mb in the 4q35.1 region. Until now, 10 patients with duplicated 4p14-p15 and deleted 4q35 chromosome 4 have been described. In all cases the abnormal chromosome 4 was derived from a pericentric inversion inherited from one of the parents. In conclusion, we have identified the first case of inv dup del(4) with normal parents suggesting that, often, terminal duplications or terminal deletions mask complex rearrangements.     

An idic(15) associated with POF (premature ovarian failure): molecular cytogenetic definition of a case and review of the literature

We report on a 36-year-old infertile woman, presenting a premature ovarian failure with an otherwise normal female phenotype. Cytogenetic analyses showed the presence of a supernumerary marker chromosome, that was characterized by FISH (fluorescent in situ hybridization) and array CGH (comparative genomic hybridization). This marker chromosome was derived from chromosome 15, and contained only heterochromatic material. The Prader Willi/Angelman region was not present. No duplications of the 15q regions were detected by array CGH. Supernumerary markers of chromosome 15 have been reported in cases of infertility and amenorrhea, that is also described in cases with marker derived by other acrocentric chromosomes. The case here presented constitutes a further example that etiology of POF is not always associated with a defective gene, but in some cases oocytes atresia can be the consequence of the abnormal meiotic pairing of chromosomes.     

分子标记技术在入侵生态学研究中的应用

外来入侵物种给农林业生产造成了巨大的经济损失,并威胁生物多样性及人类健康．入侵生态学的研究对于认识外来入侵物种的入侵机制及其可持续控制具有重要意义．分子标记技术为解决入侵生态学研究中的许多基本问题提供了良好的手段．本文综述了该技术在外来入侵物种的鉴定、地理分布、原产地、传播模式、种群遗传变异、杂交及基因渗入等研究中的应用现状,并对其应用前景进行了展望．     

Mosaic down syndrome with a marker: molecular cytogenetic characterization of the marker chromosome

Down syndrome is a complex disorder characterized by well defined and distinctive phenotypic features. Approximately 2-3% of all live-born Down individuals are mosaics. Here we report a boy with suspected Down syndrome showing mosaicism for two different cell lines where one cell line is unexpected. The cytogenetic analysis by G-banding revealed a karyotype of 47 XY+21 [20]/46,X+marker [30]. Further, molecular cytogenetic analysis with spectral karyotyping identified the marker as a derivative of Y chromosome. The delineation of Y chromosomal DNA was done by quantitative real-time PCR and aneuploidy detection by quantitative fluorescence PCR. The Y-short tandem repeats typing was performed to estimate the variation in quantity as well as to find out the extent of deletion on Y chromosome using STR markers. Fluorescence in situ hybridization using Y centromeric probe was also performed to confirm the origin of the Y marker. Further fine mapping of the marker was carried out with three bacterial artificial chromosome clones RP11-20H21, RP11-375P13, RP11-71M14, which defined the hypothetical position of the deletion. In our study we defined the extent of deletion of the marker chromosome and also discussed it in relation with mosaicism. This is the first report of mosaic Down syndrome combined with a second de novo mosaic marker derived from the Y chromosome.     

47,X,idic(Y),inv dup(Y): a non-mosaic case of a phenotypically normal boy with two different Y isochromosomes and neocentromere formation

A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)(n). The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.     

The chromosomes of Rhynchosciara baschanti (Diptera: Sciaridae): molecular cytogenetic comparisons with taxa in the americana-like group.

Polytene chromosome analysis is presented for Rhynchosciara baschanti, a species belonging to the americana-like group of Rhynchosciara. R. baschanti chromosomes show morphological differences in centromeric and telomeric regions compared to two other members within the group, R. americana and R. hollaenderi. In addition, fixed band and autosomal inversion differences were noted. Physical mapping data showed synteny among the taxa under study for DNA puffs and single-copy or histone gene probes, whereas rDNA and poly-(r)A probes showed different diagnostic patterns. The activity of developmentally active genes and the pattern of thymidine incorporation into DNA puff sites of R. baschanti are consistent with those found in the two previously studied species, except for lower levels of expression at some of these sites. These results suggest that differential duplication of specific DNA sequences, in particular repetitive and homopolymeric DNA, has played a role in the chromosomal evolution of these Rhynchosciara species. Inversions and band dimorphisms have also occurred, but the processes leading to their maintenance and fixation appear to have been slow, since these three species are in general chromosomally monomorphic.     

Characterization of an analphoid, neocentromere-positive inv dup 8p marker chromosome using multiplex whole chromosome and sub-telomere FISH analyses

A 30-year-old male patient with mild mental retardation was found to have a small supernumerary marker chromosome (SMC) in 90% of his peripheral blood cells and in 100% of his fibroblast cells. Multiplex whole chromosome and sub-telomere FISH analyses were used to determine that this SMC is an inverted duplicated distal chromosome 8p fragment. Although it was negative for alpha-DNA sequences, this marker had a functional kinetochore (neocentromere) demonstrated by a positive signal with a CENP-C antibody. Apparently intact 8p telomeres at the marker's ends were demonstrated by using a telomere repeat FISH probe. The patient's phenotypically normal mother on G-banding analysis had a small marker chromosome in 8% of her peripheral blood cells in two cultures of the first specimen studied. The marker was not seen in any subsequent maternal peripheral blood or fibroblast specimens. Although it was impossible to further characterize the maternal SMC, it was suggested that the mother had the same marker as the one seen in the proband. Inverted duplicated chromosomal fragments are the most frequent type of analphoid markers. Stable inverted duplicated 8p marker chromosomes were previously reported in three other patients. They all apparently occurred de novo and were found to be positive for kinetochore-associated proteins. Evidence for the possible inheritance of an inverted-duplicated, analphoid SMC was not shown to-date. This study also demonstrates a practical, straightforward approach for analphoid marker characterization in clinical laboratory settings, using whole chromosome multiplex and subtelomere-specific FISH analyses. FISH probes for all sub-telomere chromosomal regions are commercially available and the large majority of analphoid marker chromosomes involve telomere regions.     

Small supernumerary marker chromosomes (SMCs): genotype-phenotype correlation and classification

Small supernumerary marker chromosomes (SMCs) are present in about 0.05% of the human population. In approximately 30% of SMC carriers (excluding the ~60% SMC derived from one of the acrocentric chromosomes), an abnormal phenotype is observed. The clinical outcome of an SMC is difficult to predict as they can have different phenotypic consequences because of (1) differences in euchromatic DNA-content, (2) different degrees of mosaicism, and/or (3) uniparental disomy (UPD) of the chromosomes homologous to the SMC. Here, we present 35 SMCs, which are derived from all human chromosomes, apart from chromosome 6, as demonstrated by the appropriate molecular cytogenetic approaches, such as centromere-specific multicolor fluoresence in situ hybridization (cenM-FISH), multicolor banding (MCB), and subcentromere-specific multicolor FISH (subcenM-FISH). In nine cases without an aberrant phenotype, neither partial proximal trisomies nor UPD could be detected. Abnormal clinical findings, such as psychomotoric retardation and/or craniofacial dysmorphisms, were associated with seven of the cases in which subcentromeric single-copy probes were proven to be present in three copies. Conversely, in eight cases with a normal phenotype, proximal euchromatic material was detected as partial trisomy. UPD was studied in 12 cases and subsequently detected in two of the cases with SMC (partial UPD 4p and maternal UPD 22 in a der(22)-syndrome patient), indicating that SMC carriers have an enhanced risk for UPD. At present, small proximal trisomies of 1p, 1q, 2p, 6p, 6q, 7q, 9p, and 12q seem to lead to clinical manifestations, whereas partial proximal trisomies of 2q, 3p, 3q, 5q, 7p, 8p, 17p, and 18p may not be associated with significant clinical symptoms. With respect to clinical outcome, a classification of SMCs is proposed that considers molecular genetic and molecular cytogenetic characteristics as demonstrated by presently available methods.Electronic database information: accession numbers and URLs for the data in this article are as follows:ENSEMBL-database, National Center for Biotechnology Information (NCBI), Genome Database (GDB), OMIM (Online Mendelian Inheritance in Man) Database,     

Quinacrine fluorescence patterns in somatic chromosomes of a t(15q15q) carrier

Summary A sporadic translocation between two homologues of chromosome 15 was identified, by means of the quinacrine mustard fluorescence technique, in a phenotypically normal female infant with ventricular septal defect. Familial studies revealed certain individual variations regarding the intensely fluorescent centromeric regions in chromosomes 3 and 13, which appeared to be transmitted from the parents to offspring.

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Zusammenfassung Eine sporadische Translokation zwischen zwei homologen Chromosomen Nr. 15 wurde mit Hilfe der Quinacrine-Mustard-Fluorescenztechnik bei einem weiblichen Säugling mit Ventrikelseptumdefekt festgestellt, der sonst phänotypisch normal war. Familienuntersuchungen ergaben gewisse individuelle Varianten der Zentromerregion in den Chromosomen 3 und 13, und es wurde eine Vererbung von den Eltern auf ihre Kinder festgestellt.Familienberatungen ergaben gewisse individuelle Variationen der intensiv fluorescierenden Zentromerregion.

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Contributions from the Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo, Japan. Supported by grants, No. 584099, and No. 92035, from the Scientific Research Fund of the Ministry of Education.