Twenty-four chromosome FISH in human IVF embryos reveals patterns of post-zygotic chromosome segregation and nuclear organisation.

Fluorescence in situ hybridisation (FISH) was first applied on in vitro fertilisation (IVF) embryos for the preimplantation genetic diagnosis of sex, then chromosome translocations and later for chromosome copy number (PGS). Because of the controversy surrounding PGS diagnostically, it has been replaced by array-based approaches; however, FISH remains a powerful tool for investigating mechanisms of both post-zygotic segregation error and nuclear organisation, especially if most or all of the chromosomes in the karyotype can be analysed. The purpose of this study was to develop and apply a 24 chromosome FISH assay to investigate chromosome-specific rates of gain and loss, nuclear organisation patterns and the veracity of the original PGS result in days 5-6 human embryos. Analysis of 17 embryos by this newly developed approach gave strong signals for all chromosomes; it revealed chromosome copy number for each human chromosome per cell for each embryo and the nuclear address of the (mostly centromeric) loci probed. As all embryos were surplus to IVF requirements for both transfer and freezing (and many had an abnormal PGS indication) expected high levels of chromosome abnormalities were seen and no single nucleus displayed a normal complement; all were mosaic. Certain patterns emerged, however, namely that chromosome loss was more common than gain and apparent mitotic non-disjunction. Moreover, the centromeric probes tended preferentially to occupy the nuclear centre. Where we had a prior day 3 biopsy PGS result, it was confirmed, in part, by 24 colour FISH in most but not all cases.

Multicolour interphase cytogenetics: 24 chromosome probes, 6 colours, 4 layers.

From the late 1980s onwards, the use of DNA probes to visualise sequences on individual chromosomes (fluorescent in-situ hybridisation - FISH) revolutionised the study of cytogenetics. Following single colour experiments, more fluorochromes were added, culminating in a 24 colour assay that could distinguish all human chromosomes. Interphase cytogenetics (the detection of chromosome copy number in interphase nuclei) soon followed, however 24 colour experiments are hampered for this application as mixing fluorochromes to produce secondary colours produces images that are not easily distinguishable from overlapping signals. This study reports the development and use of a novel protocol, new fast hybridising FISH probes, and a bespoke image capture system for the assessment of chromosome copy number in interphase nuclei. The multicolour probe sets can be used individually or in sequential hybridisation layers to assess ploidy of all 24 human chromosomes in the same nucleus. Applications of this technique are in the investigation of chromosome copy number and the assessment of nuclear organisation for a range of different cell types including human sperm, cancer cells and preimplantation embryos.

Closing in on the Rieger syndrome gene on 4q25: mapping translocation breakpoints within a 50-kb region.

Rieger syndrome (RGS) is an autosomal dominant disorder of morphogenesis affecting mainly the formation of the anterior eye chamber and of the teeth. RGS has been localized to human chromosome 4q25 by linkage to epidermal growth factor (EGF). We have constructed a detailed physical map and a YAC contig of the genomic region encompassing the EGF locus. Using FISH, several YACs could be shown to cross the breakpoint in two independent RGS patients with balanced 4q translocations. Alu- and LINE-fragmentation of a 2.4-Mb YAC generated a panel of shorter YACs ranging in size from 2.4 Mb to 75 kb. Several fragmentation YACs were subcloned in cosmids, which were mapped to specific subregions of the original YAC by hybridization to the fragmentation panel to further refine the localization of the translocation breakpoints, allowing mapping of the breakpoints to within the most-telomeric 200 kb of the original 2.4-Mb YAC. FiberFISH of cosmids located in this 200-kb region mapped the two translocation breakpoints within a 50-kb region approximately 100-150 kb centromeric to D4S193, significantly narrowing down the candidate region for RGS. The mapping data and resources reported here should facilitate the identification of a gene implicated in Rieger syndrome.

An Xp22.1-p22.2 YAC contig encompassing the disease loci for RS, KFSD, CLS, HYP and RP15: refined localization of RS.

To facilitate the positional cloning of the genes involved in retinoschisis (RS), keratosis follicularis spinulosa decalvans (KFSD), Coffin-Lowry syndrome (CLS), X-linked hypophosphatemic rickets (XLH, locus name HYP) and X-linked dominant cone-rod degeneration (locus name RP15), we have extended the molecular map of the Xp22 region. Screening of several YAC libraries allowed us to identify 156 YACs, 52 of which localize between markers DXS414 (P90) and DXS451 (kQST80H1). Analysis of their marker content facilitated the construction of a YAC contig from the region spanning (in this order): DXS414 - DXS987 - DXS207 - DXS1053 - DXS197 - DXS 43 - DXS1195 - DXS418 - DXS999 - PDHA1 - DXS7161 - DXS443 - DXS 7592 - DXS1229 - DXS365 - DXS7101 - DXS7593 - DXS1052 - DXS274 - DXS989 - DXS451. The region between DXS414 and DXS451 covers about 4.5-5 Mb. Two additional markers (DXS7593 and DXS7592) were placed in the region, thereby increasing the genetic resolution. Using the deduced marker order, the analysis of key recombinants in families segregating RS allowed us to refine the critical region for RS to 0.6 Mb, between DXS418 and DXS7161.

Expression of the human Dp 71 (apo-dystrophin-1) gene from a 760-kb DMD-YAC transferred to mouse cells.

A 760-kb YAC was constructed by homologous recombination in yeast, containing the genes located in the distal portion of the DMD gene. The YAC was introduced in mouse LA-9 cells by PEG-mediated cell fusion. One transformant accommodated an intact DMD-YAC, i.e. a full copy of the DMD internal Dp 116, Dp 71 and Dp 40 genes (apo-dystrophin-2, -1 and -3, respectively). We have studied the expression of the various gene products derived from the introduced DMD-YAC. RT-PCR revealed expression of human Dp 71 but not of Dp 116 or Dp 40. Remarkably, differences were observed in processing of the 3' region of the endogenous mouse and the human transcripts, due to different splicing of exons 71 (absent in human and present in mouse transcript) and 78 (present in human and absent in mouse transcript). The splicing pattern of the human transcript is the same as that of the major Dp 71 (apo-dystrophin-1) product in human blood. The observed splicing differences may be caused by either species-specific exon use and/or by cis-acting factors, e.g. the upstream transcript composition, because we have no evidence for endogenous Dp 71 expression.

A yeast artificial chromosome contig spanning the Charcot-Marie-Tooth disease type 1A duplication region.

A contiguous set of 43 overlapping yeast artificial chromosome (YAC) clones has been developed for the Charcot-Marie-Tooth disease type 1A (CMT1A) duplication region of chromosome 17p11.2. The contig spans approximately 2.0 Mb and can be represented in a minimum of five overlapping YACs. The YAC clones were isolated from two total human genomic YAC libraries and from YAC libraries made from rodent-human hybrid cell lines. YAC clones were isolated from the libraries by polymerase chain reaction (PCR) technique. Localization to chromosome 17p11.2 was confirmed by fluorescence in situ hybridization. Overlap between the YAC clones was detected by inter-Alu PCR amplification of the YACs and by cross hybridization of the YACs with YAC insert ends obtained by Vectorette PCR. This YAC contig is a useful resource for analyzing and mapping all the genes contained within the CMT1A duplication.

Generation and fluorescent in situ hybridization mapping of yeast artificial chromosomes of 1p, 17p, 17q, and 19q from a hybrid cell line by high-density screening of an amplified library.

A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe (Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.