Rubinstein-Taybi syndrome caused by submicroscopic deletions within 16p13.3

The Rubinstein-Taybi syndrome (RTS) is a well-defined complex of congenital malformations characterized by facial abnormalities, broad thumbs and big toes, and mental retardation. The breakpoint of two distinct reciprocal translocations occurring in patients with a clinical diagnosis of RTS was located to the same interval on chromosome 16, between the cosmids N2 and RT1, in band 16p13.3. By using two-color fluorescence in situ hybridization, the signal from RT1 was found to be missing from one chromosome 16 in 6 of 24 patients with RTS. The parents of five of these patients did not show a deletion of RT1, indicating a de novo rearrangement. RTS is caused by submicroscopic interstitial deletions within 16pl3.3 in approximately 25% of the patients. The detection of microdeletions will allow the objective confirmation of the clinical diagnosis in new patients and provides an excellent tool for the isolation of the gene causally related to the syndrome.     

Genetic analysis of meiotic recombination in humans by use of sperm typing: reduced recombination within a heterozygous paracentric inversion of chromosome 9q32-q34.3.

To investigate patterns of genetic recombination within a heterozygous paracentric inversion of chromosome 9 (46XY inv[9] [q32q34.3]), we performed sperm typing using a series of polymorphic microsatellite markers spanning the inversion region. For comparison, two donors with cytogenetically normal chromosomes 9, one of whom was heterozygous for a pericentric chromosome 2 inversion (46XY inv[2] [p11q13]), were also tested. Linkage analysis was performed by use of the multilocus linkage-analysis program SPERM, and also CRI-MAP, which was adapted for sperm-typing data. Analysis of the controls generated a marker order in agreement with previously published data and revealed no significant interchromosomal effects of the inv(2) on recombination on chromosome 9. FISH employing cosmids containing appropriate chromosome 9 markers was used to localize the inversion breakpoint of inv(9). Analysis of inv(9) sperm was performed by use of a set of microsatellite markers that mapped centromeric to, telomeric to, and within the inversion breakpoints. Three distinct patterns of recombination across the region were observed. Proximal to the centromeric breakpoint, recombination was similar to normal levels. Distal to the telomeric breakpoint, there was an increase in recombination found in the inversion patient. Finally, within the inversion, recombination was dramatically reduced, but several apparent double recombinants were found. A putative model explaining these data is proposed.     

Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization

A rapid method for localizing large numbers of complete cosmids by nonradioactive in situ hybridization is described. The cosmids are nick translated in the presence of biotin-16-dUTP, incubated with an excess of sonicated human DNA, and used as a probe for in situ hybridization. Sites of hybridization are detected by successive treatments with FITC-labeled avidin and biotinylated anti-avidin antibody. Fifty-two cosmids were localized on chromosome 16 in 5 d relative to translocation breakpoints contained in two cell lines. Rapid identification of chromosome 16 was achieved by cohybridization with a chromosome 16-specific centromeric repeat probe.     

Isolation and mapping of cosmid markers on human chromosome 22, including one within the submicroscopically deleted region of DiGeorge syndrome

A genomic cosmid library was constructed from a Chinese hamster/human hybrid cell containing human intact chromosome 22 as its only human component. Of 1000 cosmids with inserts derived from human chromosome 22, 191 were tested for restriction fragment length polymorphisms (RFLPs). As a result, 64 clones detected RFLPs, including five variable number of tandem repeats systems. Of the remaining 127 cosmids, 111 detected a single copy sequence on human chromosome 22. Five somatic cell hybrids allowed us to assign all of the 64 polymorphic cosmids and 44 non-polymorphic cosmids to four different regions of human chromosome 22. In two patients with DiGeorge syndrome, one of the cosmids that had been sublocalized to 22pter-q11 detected hemizygosity. These 108 cosmid markers regionally assigned to human chromosome 22 should be useful for the construction of long-range physical maps and the identification of genetic alterations on the chromosome.     

Identification and characterization of a novel gene disrupted by a pericentric inversion inv(4)(p13.1q21.1) in a family with cleft lip

Cleft lip with or without cleft palate is a common birth defect affecting 1 in every 700 live births. Several genetic loci are believed to be involved in the pathogenesis of syndromic and non-syndromic clefting. We identified a pericentric inversion of chromosome 4, inv(4)(p13q21) that segregates with cleft lip in a two-generation family. By using a combination of fluorescence in situ hybridization, yeast artificial chromosome, bacterial artificial chromosome contig mapping, and database searching we mapped and sequenced the inversion breakpoint region. The pericentric inversion disrupts a gene (ACOD4) on chromosome 4q21 that codes for a novel acyl-CoA desaturase enzyme. The 3.0 kb human ACOD4 cDNA spans approximately 170 kb and is composed of five exons of ACOD4. The inversion breakpoint is located in the second exon. The 3.0 kb mRNA is expressed at high level in fetal brain; a lower expression level was found in fetal kidney. No expression of ACOD4 was detected in fetal lung or liver or in adult tissues. The five exons code for a protein of 330 amino acids, with a predicted molecular weight of 37.5 kDa. The protein is highly similar to acyl-CoA desaturases from Drosophila melanogaster to Homo sapiens. The catalytically essential histidine clusters and the potential transmembrane domains are well conserved.     

Detection of a cryptic paracentric inversion within band 11p13 in familial aniridia by fluorescence in situ hybridization

We report the first familial case of dominantly inherited aniridia with a cryptic inversion within band 11p13. High-resolution chromosome analysis gave a suspicion of a tiny constitutional aberration around band 11p13 and fluorescence in situ hybridization using 11p cosmids successfully confirmed that the aniridia patients of this family have an inversion within band 11p13. The distal breakpoint of the inversion is telomeric to a candidate aniridia gene (AN2) and suggests that more genes might be involved in the etiology of aniridia. In situ hybridization is a powerful tool to detect cryptic rearrangements in sporadic or familial patients with aniridia. This family indicated the importance of careful observation of the 11p13 region of aniridia patients, even if the aniridia was autosomal dominantly inherited.     

Evaluation of a cosmid contig physical map of human chromosome 16.

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.     

Localization and characterization of X chromosome inversion breakpoints separating Drosophila mojavensis and Drosophila arizonae

Ectopic exchange between transposable elements or other repetitive sequences along a chromosome can produce chromosomal inversions. As a result, genome sequence studies typically find sequence similarity between corresponding inversion breakpoint regions. Here, we identify and investigate the breakpoint regions of the X chromosome inversion distinguishing Drosophila mojavensis and Drosophila arizonae. We localize one inversion breakpoint to 13.7 kb and localize the other to a 1-Mb interval. Using this localization and assuming microsynteny between Drosophila melanogaster and D. arizonae, we pinpoint likely positions of the inversion breakpoints to windows of less than 3000 bp. These breakpoints define the size of the inversion to approximately 11 Mb. However, in contrast to many other studies, we fail to find significant sequence similarity between the 2 breakpoint regions. The localization of these inversion breakpoints will facilitate future genetic and molecular evolutionary studies in this species group, an emerging model system for ecological genetics.     

The evolutionary history of human chromosome 7

We report on a comparative molecular cytogenetic and in silico study on evolutionary changes in human chromosome 7 homologs in all major primate lineages. The ancestral mammalian homologs comprise two chromosomes (7a and 7b/16p) and are conserved in carnivores. The subchromosomal organization of the ancestral primate segment 7a shared by a lemur and higher Old World monkeys is the result of a paracentric inversion. The ancestral higher primate chromosome form was then derived by a fission of 7b/16p, followed by a centric fusion of 7a/7b as observed in the orangutan. In hominoids two further inversions with four distinct breakpoints were described in detail: the pericentric inversion in the human/African ape ancestor and the paracentric inversion in the common ancestor of human and chimpanzee. FISH analysis employing BAC probes confined the 7p22.1 breakpoint of the pericentric inversion to 6.8 Mb on the human reference sequence map and the 7q22.1 breakpoint to 97.1 Mb. For the paracentric inversion the breakpoints were found in 7q11.23 between 76.1 and 76.3 Mb and in 7q22.1 at 101.9 Mb. All four breakpoints were flanked by large segmental duplications. Hybridization patterns of breakpoint-flanking BACs and the distribution of duplicons suggest their presence before the origin of both inversions. We propose a scenario by which segmental duplications may have been the cause rather than the result of these chromosome rearrangements.     

New Molecular Markers for the Distal End of the T-Complex and Their Relationships to Mutations Affecting Mouse Development

Many mutations affecting mouse development have been mapped to the t-complex of mouse chromosome 17. We have obtained 17 cosmid clones as molecular markers for this region by screening a hamster-mouse chromosome 17 and 18 cell hybrid cosmid library with mouse-specific repetitive elements and mapping positive clones via t-haplotype vs. C3H restriction fragment length polymorphism (RFLP) analysis. Twelve of the clones mapping distal to Leh66B in t-haplotypes are described here. Using standard RFLP analysis or simple sequence length polymorphism between t-haplotypes, exceptional partial t-haplotypes and nested sets of inter-t-haplotype recombinants, five cosmids have been mapped in or around In(17)3 and seven in the most distal inversion In17(4). More precise mapping of four of the cosmids from In(17)4 shows that they will be useful in the molecular identification of some of the recessive lethals mapped to the t-complex: two cosmids map between H-2K and Crya-1, setting a distal limit in t-haplotypes for the position of the tw5 lethal, one is inseparable from the tw12 lethal, and one maps distal to tf near the t0(t6) lethal and cld.     

Assembly and analysis of cosmid contigs in the CEA-gene family region of human chromosome 19.

The carcinoembryonic antigen (CEA)-like genes are members of a large gene family which is part of the immunoglobulin superfamily. The CEA family is divided into two major subgroups, the CEA-subgroup and the pregnancy-specific glycoprotein (PSG)-subgroup. In the course of an effort to develop a set of overlapping cosmids spanning human chromosome 19, we identified 245 cosmids in a human chromosome 19 cosmid library (6-7X redundant) by hybridization with an IgC-like domain fragment of the CEA gene. A fluorescence-based restriction enzyme digest fingerprinting strategy was used to assemble 212 probe-positive cosmids, along with 115 additional cosmids from a collection of approximately 8,000 randomly selected cosmids, into five contigs. Two of the contigs contain CEA-subgroup genes while the remaining three contigs contain PSG-subgroup genes. These five contigs range in size from 100 kb to over 300 kb and span an estimated 1 Mb. The CEA-like gene family was determined by fluorescence in situ hybridization to map in the q13.1-q13.2 region of human chromosome 19. Analysis of the two CEA-subgroup contigs provided verification of the contig assembly strategy and insight into the organization of 9 CEA-subgroup genes.     

Cosmid walking and chromosome jumping in the region of PKD1 reveal a locus duplication and three CpG islands.

The locus responsible for the most common form of autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p13.3. Genetic mapping studies indicate that PKD1 is flanked on the proximal side by the DNA marker 26.6 (D16S125). Here we show that 26.6 has undergone a locus duplication and that the two loci are less than 150kb apart. One of the two loci contains a polymorphic TaqI site that has been used in genetic studies and represents the proximal boundary for the PKD1 locus. We demonstrate that the polymorphic locus is the more proximal of the two 26.6-hybridizing loci. Therefore, four cosmids isolated from the distal 26.6-hybridizing locus contain candidate sequences for the PKD1 gene. These cosmids were found to contain two CpG islands that are likely markers for transcribed regions. A third CpG island was detected and cloned by directional chromosome jumping.     

Molecular characterization of the pericentric inversion of chimpanzee chromosome 11 homologous to human chromosome 9

In addition to the fusion of human chromosome 2, nine pericentric inversions are the most conspicuous karyotype differences between humans and chimpanzees. In this study we identified the breakpoint regions of the pericentric inversion of chimpanzee chromosome 11 (PTR 11) homologous to human chromosome 9 (HSA 9). The break in homology between PTR 11p and HSA 9p12 maps to pericentromeric segmental duplications, whereas the breakpoint region orthologous to 9q21.33 is located in intergenic single-copy sequences. Close to the inversion breakpoint in PTR 11q, large blocks of alpha satellites are located, which indicate the presence of the centromere. Since G-banding analysis and the comparative BAC analyses performed in this study imply that the inversion breaks occurred in the region homologous to HSA 9q21.33 and 9p12, but not within the centromere, the structure of PTR 11 cannot be explained by a single pericentric inversion. In addition to this pericentric inversion of PTR 11, further events like centromere repositioning or a second smaller inversion must be assumed to explain the structure of PTR 11 compared with HSA 9.     

Evaluation of the genomic extent of effects of fixed inversion differences on intraspecific variation and interspecific gene flow in Drosophila pseudoobscura and D. persimilis

There is increasing evidence that chromosomal inversions may facilitate the formation or persistence of new species by allowing genetic factors conferring species-specific adaptations or reproductive isolation to be inherited together and by reducing or eliminating introgression. However, the genomic domain of influence of the inverted regions on introgression has not been carefully studied. Here, we present a detailed study on the consequences that distance from inversion breakpoints has had on the inferred level of gene flow and divergence between Drosophila pseudoobscura and D. persimilis. We identified the locations of the inversion breakpoints distinguishing D. pseudoobscura and D. persimilis in chromosomes 2, XR, and XL. Population genetic data were collected at specific distances from the inversion breakpoints of the second chromosome and at two loci inside the XR and XL inverted regions. For loci outside the inverted regions, we found that distance from the nearest inversion breakpoint had a significant effect on several measures of divergence and gene flow between D. pseudoobscura and D. persimilis. The data fitted a logarithmic relationship, showing that the suppression of crossovers in inversion heterozygotes also extends to loci located outside the inversion but close to it (within 1-2 Mb). Further, we detected a significant reduction in nucleotide variation inside the inverted second chromosome region of D. persimilis and near one breakpoint, consistent with a scenario in which this inversion arose and was fixed in this species by natural selection.     

The quantitative analysis of polymorphism on human chromosomes 1, 9, 16, and Y

Summary A paracentric inversion of chromosome 5 was detected after RHG banding in a subject affected by Klinefelter's syndrome. The inversion was also observed in the patient's mother, and was confirmed by QFQ-and RBA-banding techniques.A second paracentric inversion affecting chromosome 7 was detected in a woman with Turner's syndrome. The same structural anomaly was found in her father and her half-brother.The possible relationship between sex chromosome nondisjunction and paracentric inversion is discussed.Furthermore, the inversion of chromosome 7 reproduces exactly the chromosome 7 of the gorilla, which is presumed to be ancestral to the human 7. This therefore appears to be the first reported case of reverse chromosomal mutation.     

Unique genomic sequences in human chromosome 16p are conserved in the great apes

In humans, acute myelomonocytic leukemia (AMML) with abnormal bone marrow eosinophilia is diagnosed by the presence of a pericentric inversion in chromosome 16, involving breakpoints p13;q23 [i.e., inv(16)(p13;q23)]. A pericentric inversion involves breaks that have occurred on the p and q arms and the segment in between is rotated 180° and reattaches. The recent development of a “human micro-coatasome” painting probe for 16p contains unique DNA sequences that fluorescently label only the short arm of chromosome 16, which facilitates the identification of such inversions and represents an ideal tool for analyzing the “divergence/convergence” of the equivalent human chromosome 16 (PTR 18, GGO 17 and PPY 19) in the great apes, chimpanzee, gorilla and orangutan. When the probe is used on the type of pericentric inversion characteristic of AMML, signals are observed on the proximal portions (the regions closest to the centromere) of the long and short arms of chromosome 16. The probe hybridized to only the short arm of all three ape chromosomes and signals were not observed on the long arms, suggesting that a pericentric inversion similar to that seen in AMML has not occurred in any of these great apes. Received: 4 July 1996 / Accepted: 18 September 1996     

双色FISH和DNA纤维FISH方法的建立与应用

在构建了含毛细胞白血病相关的结构性倒位inv (5) (p13.1q13.3)的细胞系后,为了确定该新建细胞系在建株过程中其倒位断裂点关键区遗传物质是否发生改变,以生物素或地高辛标记的cCI5-216 和cCI5-267黏粒DNA为探针,进行染色体中期、间期和DNA纤维3种双色荧光原位杂交的分析。结果表明：该新建细胞系的3种双色荧光原位杂交结果,均与该细胞系的原代细胞的完全相同,证实了该细胞系倒位断裂点关键区的遗传物质结构未发生改变。该细胞系是揭示毛细胞白血病发病的分子机理的重要研究材料。