Identification of the chromosome 14 origin of a C-negative marker associated with a 14q32 deletion by chromosome painting

A constitutional chromosome 14 rearrangement was observed in a female with a psychodevelopmental disorder. Karyotype analysis using a variety of chromosome techniques, QFQ, GTG, CBG, Ag-NOR and DA-DAPI, showed a deletion of chromosome 14q32.1-qter region in association with a supernumerary marker chromosome. The marker, resembling a submetacentric, approximately half the size of a G group chromosome is C band and Ag-NOR negative. The heteromorphism of the satellites showed that the deleted chromosome 14 is paternal in origin. Chromosome painting using an Alu-PCR probe specific for the human chromosome 14 and fluorescent in situ hybridization (FISH) showed that the marker contains chromosome 14q32 sequences. It is likely that the marker was generated from the deleted chromosome 14 region through a complex rearrangement.     

Identification of complex chromosome rearrangements in the gibbon by fluorescent in situ hybridization (FISH) of a human chromosome 2q specific microlibrary, yeast artificial chromosomes, and reciprocal chromosome painting

Chromosome painting has revealed that the human chromosome homologs in lesser apes are often fragmented and translocated to a number of different hylobatid chromosomes. We investigated the fragmented human chromosome 2 homologs in gibbons to illustrate a new strategy in mapping regional and band-specific chromosomal homologies between species. Previous research showed that the DNA library specific to human chromosome 2 paints parts of four gibbon (lar species group) chromosomes (viz., 1, 10, 12, and 16) and yields five distinct hybridization signals (including two on gibbon chromosome 16). However, the exact segments of human chromosome 2 that were translocated to the various gibbon chromosomes could not be distinguished. To determine the origin of the human chromosome 2 signals, we hybridized a microlibrary for the long arm of human chromosome 2, as well as YACs specific for most of the major bands on this chromosome, to metaphases of the gibbon. For reciprocal chromosome painting, we hybridized flow-sorted gibbon chromosome probes to human chromosome 2. Each method added additional insights that helped clarify the shuffling of human chromosome 2 material in the highly reorganized gibbon genome. There was an excellent correspondence between these complementary techniques. YAC 958d2 identified the breakpoint between human chromosome 2 material present on gibbon chromosomes 10 and 16. The reciprocal chromosome painting permitted a more complete and regional assignment of homology between segments on various gibbon chromosomes to human chromosome 2. The results show that a combination of reciprocal chromosome painting, subregional microlibraries, and band-specific probes (such as YACs) can be used to identify homologies between species and to rapidly construct detailed comparative chromosome maps, especially when the karyotypes are highly rearranged.     

A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silene dioica

The segregation pattern of an 810-bp random amplified polymorphic DNA (RAPD) band in the F1 and backcross generations of a Silene dioica (L.) Clairv. family provides evidence that this molecular marker is located in the pseudoautosomal region (PAR) of the X and Y chromosomes. The marker was found through a combination of bulked segregant analysis (BSA) and RAPD techniques. Recombination rates between this pseudoautosomal marker and the differentiating portion of the Y chromosome are 15% in both generations. Alternative explanations involving nondisjunction or autosomal inheritance are presented and discussed. Chromosome counts provide evidence against the nondisjunction hypothesis, and probability calculations argue against the possibility of autosomal inheritance. This constitutes the first report of a pseudoautosomal DNA marker for plant sex chromosomes.     

Molecular typing of Salmonella typhi strains from Dhaka (Bangladesh) and development of DNA probes identifying plasmid-encoded multidrug-resistant isolates

Seventy-eight Salmonella typhi strains isolated in 1994 and 1995 from patients living in Dhaka, Bangladesh, were subjected to phage typing, ribotyping, IS200 fingerprinting, and PCR fingerprinting. The collection displayed a high degree of genetic homogeneity, because restricted numbers of phage types and DNA fingerprints were observed. A significant number of the S. typhi strains (67%) were demonstrated to be multiple drug resistant (MDR). The vast majority of the MDR strains were resistant to chloramphenicol, ampicillin, trimethoprim, streptomycin, sulfamethoxazole, and tetracycline (R type CATmSSuT), a resistance phenotype that has also frequently been observed in India. Only two strains displayed a distinct MDR phenotype, R type AT-mSSuT. Pulsed-field gel electrophoresis demonstrated the presence of large plasmids exclusively in the MDR strains of both R types. The plasmids present in the S. typhi strains of R type CATmSSuT could be conjugated to Escherichia coli and resulted in the complete transfer of the MDR phenotype. PCR fingerprinting allowed discrimination of MDR and susceptible strains. The DNA fragments enabling discrimination of MDR and susceptible S. typhi strains by PCR were useful genetic markers for identifying MDR encoded by large plasmids of the H1 incompatibility group.     

Mosaicism for a small supernumerary ring X chromosome in a dysmorphic, growth-retarded male: mos47,XXY/48,XXY, +r(X)

Supernumerary ring X [r(X)] chromosomes are often found in patients with Turner syndrome. The phenotypic effects of the r(X) chromosome are variable, and largely depend on the presence or absence of the X inactivation (XIST) locus. Ring(X) chromosomes in males are rare and have been previously reported in only four cases, with 47,XY, + r(X) or mos47,XY, +r(X)/46,XY karyotypes. These patients all had developmental delay and dysmorphic features. We describe a 2.5-year-old male patient with facial dysmorphia, growth retardation, microcephaly, global developmental delay, and microphallus. Cytogenetic analysis from peripheral blood lymphocytes and fibroblasts identified mosaicism for two cell lines: mos48,XXY, + r(?X)/47,XXY. Fluorescence in situ hybridization (FISH) with an X chromosome paint showed the ring chromosome to be X chromosome derived. This is the first case of an r(X) chromosome described in a 47,XXY patient. FISH analysis of the r(X) chromosome with an XIST probe showed that the XIST locus was absent. Functional disomy of genes in the r(X) chromosome most likely accounts for the abnormal phenotype in the proband.     

Evaluation of X, Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization

Polymorphonuclear leukocytes (PMNs) isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis and from peripheral blood of volunteers were stimulated with 12-phorbol-13-myristate acetate (PMA). No significant differences in luminol-amplified chemiluminescence were found between different patients and control groups. However, two distinct patterns of native chemiluminescence were observed. Type I showed no, or only a small, increase in native chemiluminescence with integral counts over 30 min less than 3 x 10(5) cpm, and the majority of samples from volunteers were of this type. Type II was characterized by a burst of native chemiluminescence starting 8 to 15 min after cell stimulation. It was found in most PMN samples from patients with rheumatoid arthritis. Integral counts over 30 min were always higher than 10(6) cpm and as high as 10(8) cpm in some cases. A strong inhibition of the Type II native chemiluminescence was caused by desferal, catalase, thiourea, and glutathione. However, the luminol-amplified chemiluminescence remained unchanged or was only slightly decreased under the same experimental conditions. Sodium azide strongly inhibited both kinds of luminescence. Hydroxyl radicals, formed in a Fenton reaction, may be important intermediates in the Type II native chemiluminescence.     

Pilot studies for proficiency testing using fluorescence in situ hybridization with chromosome-specific DNA probes: a College of American Pathologists/American College of Medical Genetics Program

This paper proposes that a comprehensive, long-term program with a case-management focus will produce better outcomes and be more cost-effective than the current approach to managing the illnesses of women on Temporary Assistance for Needy Families (or TANF, formerly known as AFDC) who are afflicted with both drug dependency and mental illness, i.e. a dual diagnosis. It is proposed that this comprehensive approach would diminish the generational cycle of substance abuse, dysfunction (including violence), and dependence on public support, which is too often the pattern in single-parent homes where the mother has been dually diagnosed. For our purposes, dual diagnosis is defined as any mental health diagnosis using the DSM-IV criteria coexisting with a diagnosis of substance abuse, whether licit or illicit. Current drug policy, particularly as it applies to those with a dual diagnosis, has an emphasis on criminal justice system solutions. It is extremely expensive (incarceration alone is variously estimated as costing $25,000 to $45,000 per year per person), and does little to treat, prevent, or consequently, reduce the problem. The model design discussed in this article provides for comprehensive treatment and support services to women with a dual diagnosis receiving TANF. Its goal is to help break the family cycle of system dependency. The article hypothesizes that if a well-designed program evaluation is implemented, it will demonstrate savings in reduced health care, criminal justice, and social service costs.     

Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection

To evaluate the role of excitatory amino acids in secondary injury occurring after spinal cord trauma, several experimental studies focusing on the the changes of amino acid levels in the spinal cord have been performed to date. However, because of technical limitations, it has not been possible to correlate the local changes of excitatory amino acids with the total tissue levels of excitatory amino acids. To investigate the connection between the spread of injury and the excitatory amino acids, we assessed, the local changes of aspartate through novel experimental approaches like immunoreactivity via fluorescence microphotometry and histopathology while also analyzing the total tissue levels of amino acids via HPLC. These studies were performed using a model of incomplete cervical spinal cord injury in rats. Through this approach, we found that the levels of excitatory amino acids, such as glutamate and aspartate, began to decrease immediately after injury. No significant decrease was observed in the other amino acids. Similarly, local changes in aspartate in the spinal cord were observed using fluorescence microphotometry. The decrease in the anterior and posterior horns was rapid up to 15 min after injury, but, slowed thereafter, suggesting that a release of excitatory amino acids occurred at the site of primary injury almost immediately following injury. At 15-min post-injury large neurons within the injured cord appeared intact on histopathological analysis demonstrating that the alteration of excitatory amino acids occurs prior to histopathological change. Histopathological change in the white matter occurred more slowly than in the anterior and posterior horns, suggesting the spread of the lesion by secondary damage due to an autoclastic mechanism.     

Evidence by allelic association-dependent methods for a type 1 diabetes polygene (IDDM6) on chromosome 18q21

Type 1 diabetes is a common polygenic disease. Fine mapping of polygenes by affected sibpair linkage analysis is not practical and allelic association or linkage disequilibrium mapping will have to be employed to attempt to detect founder chromosomes. Given prior evidence of linkage of the Jk-D18S64 region of chromosome 18q12-q21 to type 1 diabetes, we evaluated the 12 informative microsatellite markers in the region for linkage with disease by the transmission disequilibrium test (TDT) in a UK data set of type 1 diabetic families (n = 195). Increased transmission of allele 4 of marker D18S487 to affected children was detected (P = 0.02). Support for this was extended in a total of 1067 families from four different countries by isolating, and evaluating by the TDT, two novel microsatellites within 70 kb of D18S487. Evidence for linkage and association was P = 5 x 10(-5) and 3 x 10(-4), respectively. There was no evidence for increased transmission of associated alleles to nonaffected siblings. Analysis of an additional 390 families by the TDT did not extend the evidence further, and reduced support in the total 1457 families to P = 0.001 for linkage and P = 0.003 for association. However, evidence for linkage by affected sibpair allele sharing was strong (P = 3.2 x 10(-5)) in the second data set. Heterogeneity in TDT results between data sets was, in part, accounted for by the presence of more than one common disease-associated haplotype (allelic heterogeneity) which confounds the analysis of individual alleles by the TDT. Guidelines for strategies for the mapping of polygenes are suggested with the emphasis on collections of large numbers of families from multiple populations that should be as genetically homogeneous as possible.     

Prenatal diagnosis of supernumerary chromosome derivative (22) due to maternal balanced translocation in association with diaphragmatic hernia: a case report

An aneuploid fetus was detected prenatally by cordocentesis at 27 weeks' gestation following ultrasonographic diagnosis of severe fetal growth retardation and a large diaphragmatic hernia. The fetal karyotype was revealed to be 47,XX,der(22)t(11;22)(q23.3;q11.2) after parental bloods confirmed a balanced reciprocal translocation in the mother. Approximately 85 cases with an unbalanced karyotype 47,XX(or XY),+der(22),t(11;22) due to 3:1 meiotic disjunction in the parental translocation carrier have been reported in the world literature and only one of them was diagnosed prenatally. This is the first detailed case report of a supernumerary derivative (22) chromosome abnormality diagnosed prenatally in association with diaphragmatic hernia.     

Rapid cloning of mouse DNA as yeast artificial chromosomes by transformation-associated recombination (TAR)

The purpose of this study is to investigate the count-characteristics of wrist actigraphy in basic human activities and to discuss the agreement of sleep-wake identification between polysomnography (PSG) and wrist actigraphy during nocturnal sleep. There was a distinct distribution of actigraphy counts over the studied activities. The evaluation of sleep-wake scoring using the wrist actigraphy agreed 96.9% with the polysomnographic scoring during nocturnal sleep.     

Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences

A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human beta-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence in situ hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.     

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.     

Clinical relevance of the i(12p) marker chromosome in germ cell tumors

BACKGROUND: Germ cell tumors in men are curable at all stages and are among the most sensitive of all cancers to chemotherapy. An isochromosome of the short arm of chromosome 12, i(12p), has been reported to be a frequent marker of these tumors and to have diagnostic and prognostic significance. PURPOSE: We evaluated the possible association between this cytogenetic marker and clinical outcome for men with germ cell tumors. METHODS: One hundred seventy-eight germ cell tumor samples from 150 men were studied using conventional and molecular cytogenetic techniques. Of these samples, 171 were evaluable. Patient characteristics, disease stage, treatment outcome, and disease status were correlated with the observed cytogenetic changes. In addition, 28 biopsy specimens obtained from 28 patients with tumors of uncertain histogenesis were evaluated to determine whether the presence of i(12p) could serve as a diagnostic marker of a germ cell origin for these tumors. RESULTS: Of the 171 evaluable tumor accessions, 101 (59%) yielded abnormal karyotypes. i(12p) was determined to be present in 79 of the 101 (79%) abnormal karyotypes, which were derived from all cell types and primary sites. An abnormal karyotype was more frequently obtained from nonseminomatous tumors (91/137 [81%]) than from seminomas (10/34 [30%] [P < .001]). Tumors resulting in a cytogenetic failure were more likely to respond completely to chemotherapy than tumors with an abnormal karyotype (P = .004). i(12)p copy number was not associated with response or survival. Fluorescence in situ hybridization using a chromosome 12 centromere-specific probe detected i(12p) in 47 of 47 tumors (100%) already shown to have i(12p) by cytogenetic analysis and in 13 of 49 tumors (27%) exhibiting either an abnormal karyotype or a cytogenetic failure. One or more copies of i(12p), excess 12p copy number, or a deletion on the long arm of chromosome 12 was found in seven of 28 (25%) midline tumors of uncertain histogenesis, thus establishing a diagnosis of a germ cell tumor in these patients. One partial and five complete responses were observed in these seven patients. Only two partial responses were seen in the 17 patients who had no detectable germ cell tumor-related cytogenetic marker (P = .009). CONCLUSIONS: i(12p) is a highly nonrandom chromosomal marker seen in about 80% of male germ cell tumors with evaluable cytogenetic abnormalities. The presence of this isochromosome has diagnostic and possibly prognostic importance for patients with these tumors. IMPLICATIONS: Cytogenetic studies of germ cell tumors in prospective clinical treatment trials are warranted to define more precisely the relationship between histologic subtype, serum tumor marker production, and prognosis.     

Clinical applications of primed in situ labelling (PRINS) rapid identification of a marker chromosome in a fetus

This article describes the adaptation of a pattern search algorithm, for the computational optimization of model molecular structures, to run on a parallel computer. The parallel efficiency (speedup) of the algorithm is discussed, and the rate of convergence of the parallel procedure is compared to that of the sequential implementation. More generally, the article also illustrates how inherently nonparallelizable stochastic procedures may be successfully parallelized by rearrangement of the algorithm.     

Identification of the Par2 (Pulmonary adenoma resistance) locus on mouse chromosome 18, a major genetic determinant for lung carcinogen resistance in BALB/cByJ mice

The A/J mouse strain is 14 times more susceptible to urethane-induction of lung carcinogenesis than the BALB/cByJ strain (BALB). The relative resistance of BALB is dominant over the high sensitivity of A/J, since (BALBxA/J)F1 mice are phenotypically similar to the parental BALB mice. BALB mice must thus possess modifier genes suppressing phenotypic expression of the Pas (Pulmonary adenoma susceptibility) genes, which are known to be dominant genetic determinants for lung carcinogenesis in A/J mice. In order to genetically dissect the dominant resistance of the BALB mouse, we performed a linkage analysis to chromosomally map modifier genes by using 130 (A/JxBALB)F1xA/J backcross mice. Each backcross mouse was injected i.p. with urethane (1 mg/g bw) at 6 weeks of age and lung tumors were enumerated after 120 days. When the backcross mice were genotyped at multiple simple sequence repeat marker loci distributed on all the chromosomes, a significant linkage between the presence of a BALB allele and resistance to lung tumor induction was found on distal chromosome 18 (maximum LOD = 12.2). Thus, distal chromosome 18 of the BALB mouse contains a modifier gene for lung carcinogenesis: The locus, designated Par2 (Pulmonary adenoma resistance), accounted for 38% of the phenotypic variance in the backcross population, indicating a major role in protection against lung tumor development.     

Del(18p) shown to be a cryptic translocation using a multiprobe FISH assay for subtelomeric chromosome rearrangements

We have previously described a fluorescence in situ hybridisation (FISH) assay for the simultaneous analysis of all human subtelomeric regions using a single microscope slide. Here we report the use of this multiprobe FISH assay in the study of a patient whose karyotype was reported by G banding analysis as 46,XX,del(18)(p11.2). Although the proband had some features suggestive of a chromosomal abnormality, relatively few of the specific features of del(18p) were present. She was a 37 year old female with mild distal spinal muscular atrophy (SMA), arthritis of the hands, an abnormal chest shape (pectus excavatum), and an unusual skin condition (keratosis pilaris). Reverse chromosome painting with degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplified del(18p) chromosomes as a probe confirmed the abnormality as del(18p), with no evidence of any other chromosome involvement. Subsequently, the multiprobe FISH assay confirmed deletion of 18p subtelomeric sequence. However, the assay also showed that sequences corresponding to the 2p subtelomeric probe were present on the tip of the shortened 18p. The patient is therefore monosomic for 18p11.2-pter and trisomic for 2p25-pter, and the revised karyotype is 46,XX,der(18)t(2;18)(p25; p11.2). We believe that a proportion of all cases reported as telomeric deletions may be cryptic translocations involving other chromosome subtelomeric regions. Further studies such as this are necessary to define accurately the clinical characteristics associated with pure monosomy in chromosomal deletion syndromes.