Mice lacking the homologue of the human 22q11.2 gene CRKL phenocopy neurocristopathies of DiGeorge syndrome.

Heterozygous deletions within human chromosome 22q11 are the genetic basis of DiGeorge/velocardiofacial syndrome (DGS/VCFS), the most common deletion syndrome (1 in 4,000 live births) in humans. CRKL maps within the common deletion region for DGS/VCFS (ref. 2) and encodes an SH2-SH3-SH3 adapter protein closely related to the Crk gene products. Here we report that mice homozygous for a targeted null mutation at the CrkL locus (gene symbol Crkol for mice) exhibit defects in multiple cranial and cardiac neural crest derivatives including the cranial ganglia, aortic arch arteries, cardiac outflow tract, thymus, parathyroid glands and craniofacial structures. We show that the migration and early expansion of neural crest cells is unaffected in Crkol-/- embryos. These results therefore indicate an essential stage- and tissue-specific role for Crkol in the function, differentiation, and/or survival of neural crest cells during development. The similarity between the Crkol-/- phenotype and the clinical manifestations of DGS/VCFS implicate defects in CRKL-mediated signaling pathways as part of the molecular mechanism underlying this syndrome.

CpG islands of chicken are concentrated on microchromosomes.

The chicken karyotype comprises 39 chromosome pairs of which at least 29 are 'microchromosomes'. Microchromosomes account for about 25% of the genomic DNA, but they are cytologically indistinguishable from one another (1). Due to technical limitations there is a strong bias of mapped genes within the chicken genome database ChickGBASE (2) towards macrochromosomes 1-6 and Z, with specific assignments to only one microchromosome (3,4). Several genes have, however, been assigned to the microchromosome group as a whole (3,5-9), demonstrating that these tiny chromosomes do not represent genetically inert DNA. To determine the overall chromosomal distribution of genes, as well as to provide a mapping resource, we prepared a CpG island library from chicken using differential binding to a methyl-CpG chicken using differential binding to a methyl-CpG binding column before and after de novo methylation (10). Surprisingly, we found that chicken CpG islands are highly concentrated on the microchromosomes, whereas macrochromosomes 1-6 are comparatively gene-poor by this assay. Our results raise the possibility that gene density on chicken microchromosomes approaches the maximum value known for vertebrates.

FISH mapping of de novo apparently balanced chromosome rearrangements identifies characteristics associated with phenotypic abnormality.

We report fluorescence in situ hybridization (FISH) mapping of 152, mostly de novo, apparently balanced chromosomal rearrangement (ABCR) breakpoints in 76 individuals, 30 of whom had no obvious phenotypic abnormality (control group) and 46 of whom had an associated disease (case group). The aim of this study was to identify breakpoint characteristics that could discriminate between these groups and which might be of predictive value in de novo ABCR (DN-ABCR) cases detected antenatally. We found no difference in the proportion of breakpoints that interrupted a gene, although in three cases, direct interruption or deletion of known autosomal-dominant or X-linked recessive Mendelian disease genes was diagnostic. The only significant predictor of phenotypic abnormality in the group as a whole was the localization of one or both breakpoints to an R-positive (G-negative) band with estimated predictive values of 0.69 (95% CL 0.54-0.81) and 0.90 (95% CL 0.60-0.98), respectively. R-positive bands are known to contain more genes and have a higher guanine-cytosine (GC) content than do G-positive (R-negative) bands; however, whether a gene was interrupted by the breakpoint or the GC content in the 200 kB around the breakpoint had no discriminant ability. Our results suggest that the large-scale genomic context of the breakpoint has prognostic utility and that the pathological mechanism of mapping to an R-band cannot be accounted for by direct gene inactivation.

Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

We used mitotic chromosomes isolated from a human EJ bladder carcinoma cell line for morphological transformation of mouse C127 cells. These chromosome-mediated transformants were analyzed for cotransfer of markers syntenic with c-Ha-ras-1 on human chromosome 11. We also used cloned, dispersed human DNA repeats, in a general mapping strategy, to quantitate the amounts and molecular state of human DNA transferred along with the activated c-Ha-ras-1 gene. In situ hybridization was used to visualize the physical state of the transfected human chromatin. The combined use of these various techniques revealed the occurrence of both chromosomal and DNA rearrangements. However, our analysis also demonstrated that, in general, very substantial lengths of DNA are transferred intact. Closely linked markers are likely to cosegregate. Therefore, these transformants should be invaluable sources for the complete molecular cloning of isolated fragments of the short arm of human chromosome 11.

Confirmation of CHD7 as a cause of CHARGE association identified by mapping a balanced chromosome translocation in affected monozygotic twins.

BACKGROUND: CHARGE syndrome has an estimated prevalence of 1/10,000. Most cases are sporadic which led to hypotheses of a non-genetic aetiology. However, there was also evidence for a genetic cause with reports of multiplex families with presumed autosomal dominant, possible autosomal recessive inheritance and concordant twin pairs. We identified a monozygotic twin pair with CHARGE syndrome and a de novo balanced chromosome rearrangement t(8;13)(q11.2;q22). METHODS: Fluorescence in situ hybridisation was performed with BAC and PAC probes to characterise the translocation breakpoints. The breakpoint on chromosome 8 was further refined using 10 kb probes we designed and produced using sequence data for clone RP11 33I11, the Primer3 website, and a long range PCR kit. RESULTS: BAC and PAC probe hybridisation redefined the breakpoints to 8q12.2 and 13q31.1. Probe RP11 33I11 spanned the breakpoint on chromosome 8. Using our 10 kb probes we demonstrated that the chromodomain gene CHD7 was disrupted by the translocation between exons 3 and 8. DISCUSSION: Identifying that the translocation breakpoint in our patients occurred between exons 3 and 8 of CHD7 suggests that disruption of this gene is the cause of CHARGE syndrome in the twins and independently confirms the role of CHD7 in CHARGE syndrome.

Molecular characterisation of four cases of intrachromosomal triplication of chromosome 15q11-q14.

CONTEXT: Chromosomal abnormalities that involve the proximal region of chromosome 15q occur relatively frequently in the human population. However, interstitial triplications involving one 15 homologue are very rare with three cases reported to date. OBJECTIVE: To provide a detailed molecular characterisation of four additional patients with interstitial triplications of chromosome 15q11-q14. DESIGN: Molecular analyses were performed using DNA markers and probes specific for the 15q11-q14 region. SETTING: Molecular cytogenetics laboratory at the University of Chicago. SUBJECTS: Four patients with mild to severe mental retardation and features of Prader-Willi syndrome (PWS) or Angelman syndrome (AS) were referred for molecular cytogenetic analysis following identification of a suspected duplication/triplication of chromosome 15q11-q14 by routine cytogenetic analysis. MAIN OUTCOME MEASURES: Fluorescence in situ hybridisation (FISH) was performed to determine the type of chromosomal abnormality present, the extent of the abnormal region, and the orientation of the extra chromosomal segments. Molecular polymorphism analysis was performed to determine the parental origin of the abnormality. Methylation and northern blot analyses of the SNRPN gene were performed to determine the effect of extra copies of the SNRPN gene on its methylation pattern and expression. RESULTS: Fluorescence in situ hybridisation (FISH) using probes within and flanking the Prader-Willi/Angelman syndrome critical region indicated that all patients carried an intrachromosomal triplication of proximal 15q11-q14 in one of the two chromosome 15 homologues (trip(15)). In all patients the orientation of the triplicated segments was normal-inverted-normal, suggesting that a common mechanism of rearrangement may have been involved. Microsatellite analysis showed the parental origin of the trip(15) to be maternal in three cases and paternal in one case. The paternal triplication patient had features similar to PWS, one maternal triplication patient had features similar to AS, and the other two maternal triplication patients had non-specific findings including hypotonia and mental retardation. Methylation analysis at exon 1 of the SNRPN locus showed increased dosage of either the paternal or maternal bands in the paternal or maternal triplication patients, respectively, suggesting that the methylation pattern shows a dose dependent increase that correlates with the parental origin of the triplication. In addition, the expression of SNRPN was analysed by northern blotting and expression levels were consistent with dosage and parental origin of the triplication. CONCLUSIONS: These four additional cases of trip(15) will provide additional information towards understanding the phenotypic effects of this abnormality and aid in understanding the mechanism of formation of other chromosome 15 rearrangements.

Organisation of the pericentromeric region of chromosome 15: at least four partial gene copies are amplified in patients with a proximal duplication of 15q.

Clinical cytogenetic laboratories frequently identify an apparent duplication of proximal 15q that does not involve probes within the PWS/AS critical region and is not associated with any consistent phenotype. Previous mapping data placed several pseudogenes, NF1, IgH D/V, and GABRA5 in the pericentromeric region of proximal 15q. Recent studies have shown that these pseudogene sequences have increased copy numbers in subjects with apparent duplications of proximal 15q. To determine the extent of variation in a control population, we analysed NF1 and IgH D pseudogene copy number in interphase nuclei from 20 cytogenetically normal subjects by FISH. Both loci are polymorphic in controls, ranging from 1-4 signals for NF1 and 1-3 signals for IgH D. Eight subjects with apparent duplications, examined by the same method, showed significantly increased NF1 copy number (5-10 signals). IgH D copy number was also increased in 6/8 of these patients (4-9 signals). We identified a fourth pseudogene, BCL8A, which maps to the pericentromeric region and is coamplified along with the NF1 sequences. Interphase FISH ordering experiments show that IgH D lies closest to the centromere, while BCL8A is the most distal locus in this pseudogene array; the total size of the amplicon is estimated at approximately 1 Mb. The duplicated chromosome was inherited from either sex parent, indicating no parent of origin effect, and no consistent phenotype was present. FISH analysis with one or more of these probes is therefore useful in discriminating polymorphic amplification of proximal pseudogene sequences from clinically significant duplications of 15q.

Tandem duplication of 11p12-p13 in a child with borderline development delay and eye abnormalities: dose effect of the PAX6 gene product?

We report on a girl with a duplication of chromosome band 11p12-->13, which includes the Wilms tumor gene (WT1) and the aniridia gene (PAX6). The girl had borderline developmental delay, mild facial anomalies, and eye abnormalities. Eye findings were also present in most of the 11 other published cases with partial trisomy 11p, including 11p12-->13. Recently, it was shown that introduction of additional copies of the PAX6 gene into mice caused very variable eye abnormalities. Therefore, a PAX6 gene dosage effect is likely to be present in mice and humans. The central nervous system may be less sensitive to an altered PAX6 gene dosage, which is consistent with the borderline developmental delay in the present patient. Urogenital abnormalities were absent in this patient and in most of the other patients with partial trisomy of 11p. Therefore, the effect of a WT1 gene duplication on the embryological development of the urogenital tract remains uncertain.

Human chromosome analysis and sorting.

Flow cytometry has provided the cytogeneticist with a fast and accurate method of measuring the quantity of DNA in each human chromosome (1). Almost all the chromosomes in the human complement can now be resolved and abnormal chromosomes and aneuploidies (13,21, and X) recognized. A flow karyotype shows a pattern of peaks and troughs that is unique for each individual or cell line because of the variation in heterochromatic regions of the chromosomes (2). When combined with family studies, flow cytometry has been able to resolve homologues differing in DNA content by as little as 1/2000 of the human genome (3,4), less than a metaphase band. In addition, the sorting capabilities of most flow machines have provided a method for the purification of small but useful quantities of individual chromosomes, for example, 2×10(6) average sized human chromosomes are equivalent to 500 ng of DNA. Using recombinant DNA techniques, this material can be used to generate a large number of DNA probes to produce a chromosome-specific library, which can be used for the molecular analysis of genetic disease (5,6). More recently, molecular biologists have experimented with gene mapping by sorting small quantities of individual chromosomes onto filters for spot-blot hybridization with DNA probes (7).

Application of automation to the detection of radiation damage using FISH technology.

A whole chromosome painting approach was employed to highlight the damaging effects of low-to-moderate doses of ionizing radiation. A detailed tally of damage involving the painted chromosomes 1 and 2 was compiled from visual analysis and compared with the results of an automatic image processing approach, where the possible outcomes were 'normal', 'abnormal', or 'rejected'. The performance of the automatic approach was tested using a set of 9000 bicolour metaphase images harvested from whole-blood cell culture following irradiation levels of 0.0, 0.5, 1.0 and 2.0 Gy. Every metaphase image in the set was analysed visually. The automatic analysis model was based on two simple image criteria to distinguish normal from abnormal; either an increase in the number of painted objects or a large asymmetry in the area distribution of the expected number of painted objects. A result was obtained without a full karyotype analysis. In practice, automatic analysis produced a set of images for review that were enriched by a factor of 3-4 in true abnormal images. Fast visual review of these images (approximately 200/h) selected the true abnormals. A comparison of the automatic analysis with the visual analysis showed that automated analysis correctly identified 60% of normal cells, 59% of abnormal cells and 73% of rejected cells.

Detecting radiation damage to human chromosomes by flow cytometry.

The flow karyotype profile of ethidium bromide-stained chromosomes from human peripheral blood lymphocytes has been analysed following exposure of lymphocytes to graded series of X-ray doses in vitro. Flow analysis offers the potential for rapid counting of chromosome abnormalities and it is shown that the level of background fluorescence, the distribution of fluorescence and the area of peaks associated with the larger chromosomes, are altered in a dose-related fashion following previous exposures of cultured lymphocytes to 50-400 rad. Moreover, parallel manual analysis of the incidence of chromosome aberrations in metaphase samples of the irradiated cells show a close correlation between flow karyotype profile distortion and aberration frequency. It is estimated that for any given irradiated blood sample doses above 100 rad could be detected with certainty.

Karyotype and chromosomal banding pattern in Heteropeza pygmaea.

The chromosomes of somatic and germ line cells of female embryos produced by paedogenesis were studied. The haploid set in somatic cells consists of one long submetacentric chromosome, one large acrocentric, one medium metacentric and two small acrocentrics. The length vs arm index karyogram makes it possible to distinguish all but the two pairs of small acrocentric chromosomes. --Atempts were made to develope a method for banding pattern visualization. The best result was obtained using trypsin which induced banding in the chromosomes of the somatic cells and occasionally also the germ line cells. The resulting banding patterns were frequently not identical in members of a chromosome pair. There was also a variation between metaphases within an embryo as well as from different embryos. Some tentative explanations for these results are discussed.

Use of an alphoid satellite sequence to locate the X chromosome automatically, with particular reference to identification of the fragile X.

An alphoid DNA sequence primarily located on the X chromosome was labeled with biotin and hybridized in situ to preparations of metaphase chromosomes derived from fragile X-affected individuals; hybridization sites were detected immunologically. Labeled X chromosomes were located automatically in digitized images of metaphase cells by searching for the concurrence of a pronounced peak in the longitudinal density profile with the centromere in medium-sized chromosomes having a suitable centromeric index. Approximately 70% of the X chromosomes were detected by a simple classifier; this rate is similar to the automatic classification rate obtained with G-banded metaphases. The frequency of detection of the fragile X site obtained when scored directly from the microscope using this new preparation technique did not differ significantly from the frequency obtained in the same sample by means of a conventional technique. The frequency obtained by visual scoring of digitized images was slightly higher, but not significantly so.

Purifying human Y chromosomes by flow cytometry and sorting.

A method of producing an enriched sample of human Y chromosomes from peripheral blood lymphocytes is described. Metaphase chromosomes were prepared from peripheral blood lymphocytes donated by 17 normal male individuals. A suspension of chromosomes in a polyamine buffer was produced from each sample, stained with the fluorescent dye Hoechst 33258, and passed through a flow cytometer and sorter. Following analysis of the 17 fluorescence distributions, a single donor was found giving a separate peak corresponding to the Y chromosome. Seventy percent of the chromosomes sorted from this peak were identified as Y chromosomes. Batches of a million Y chromosomes were produced from each of several 40 ml donations of peripheral blood. These were assessed for the amount of Y DNA present and used to construct a DNA library.

Automatic detection of fragile X chromosomes using an X centromere probe.

In order to score for the fragile X syndrome, blood samples are prepared with absorption stain labeling by in situ hybridisation of the X chromosome centromeres. Metaphases are located, digitised at high resolution, and segmented fully automatically. A three stage adaptive classification scheme for labeled X chromosomes is then applied. This consists of a simple box classifier to identify plausible X and false positive X chromosomes, followed by a quadratic discriminant classifier that is re-trained for each sample. The modal number of X chromosomes is then determined for each sample and used to refine the classification. A simple fragile site detector is applied to the distal portion of the detected X chromosome long arms. From the results we estimate computer and operator time requirements for a screening system in which the operator reviews only the apparently fragile X chromosomes detected by the computer.

Functional reintroduction of human telomeres into mammalian cells.

Telomeric sequences of eukaryotes consist of short tandem repeats organized in arrays of variable length in which the guanine-rich strand runs 5'----3' toward the chromosomal end. The terminal repeats in yeast are the only elements necessary for telomere function in this organism. To test whether mammalian terminal repeats can function after reintroduction into a mammalian cell, a repeat-containing terminal fragment from a human chromosome was electroporated into a hamster-human hybrid cell line. In 6 of 27 independent transformants analyzed, the introduced sequences were found at the ends of chromosomes, based on all available criteria. Terminal restriction-fragment heterogeneity and the survival of these chromosomes demonstrate that these telomeres are functional. Cytogenetic evidence from one of these cell lines suggests that chromosome breakage with healing at the integration site is the mechanism responsible for the terminal location.

Lambda CM8, a human sequence with putative centromeric function, does not map to the centromere but is present in one to two copies at 9qter.

A DNA fragment isolated from a human genomic library, was reported to be present at all human centromeres and present at 16-32 copies per genome. Reintroduction of this DNA into mammalian cells as a concatenated phage clone gave rise to dicentric chromosomes which gave rise to a new, stable, chromosome. Taken together these observations could mean that this DNA is part of a native centromere. We have reexamined the location and copy number of this sequence and find it to be present at 1-2 copies per genome with a single site of in situ hybridisation at 9qter.