Chromosomal localization of the genes for the vitronectin and fibronectin receptors alpha subunits and for platelet glycoproteins IIb and IIIa.

The integrins, a family of related membrane receptors involved in cell-cell and cell-matrix interactions, are heterodimeric complexes of alpha and beta subunits. To begin to understand the evolution of these complexes, we studied the genomic organization of several alpha and beta integrin subunits. Using both somatic cell hybrids and an in situ hybridization technique, we have determined the chromosomal location of the genes for the alpha subunits of the vitronectin receptor (VNR alpha), the fibronectin receptor (FNR alpha), and for the alpha subunit of the platelet glycoprotein IIb/IIIa complex, GPIIb. In addition, we have determined the chromosomal location of the gene for the beta subunit of the GPIIb/IIIa heterodimer, GPIIIa. Our studies indicate that the alpha subunits do not localize to a single locus, but that each is found on a different chromosome. The gene for VNR alpha is located on chromosome 2, the gene for FNR alpha is on chromosome 12q11----13, and the gene for GPIIb is on chromosome 17q21----23. In contrast to the chromosomal dispersion of the alpha subunits, the genes for GPIIb and GPIIIa are physically close, with the gene for GPIIIa also located on chromosome 17q21----23. These studies indicate that the genes for the alpha subunits of the integrin family have been dispersed during evolution while GPIIb and GPIIIa are in close physical proximity. This physical proximity of GPIIb and GPIIIa may be involved in the concurrent expression of these proteins by megakaryocytes, and may result in linkage disequilibrium between these two genes, which would limit the use of restriction length polymorphisms in linkage studies of GPIIb/IIIa abnormalities in small kindreds.

Localization of myeloperoxidase to the long arm of human chromosome 17: relationship to the 15; 17 translocation of acute promyelocytic leukemia.

Myeloperoxidase (MPO) is an enzyme whose synthesis is restricted to the promyelocytic stage of myeloid differentiation. We have recently described the cloning and sequencing of a cDNA for MPO. Using a regional mapping panel of somatic cell hybrids containing various deleted or translocated segments of chromosome 17, we have assigned MPO to a region between 17q21 and 17q23. In situ hybridization refined this localization in that grains on chromosome 17 were significantly clustered at bands q22-23 and no hybridization was detected at q21. In light of this chromosome assignment, the relationship of MPO to the 17q translocation breakpoint characteristic of acute promyelocytic leukemia (APL) was considered. Because this breakpoint has been variously assigned to different bands on 17q from 17q11.2 to 17q22, the cytogenetic and molecular distance between this breakpoint and MPO cannot be accurately determined. MPO and other probes mapped to this region of 17 will be important in searching for altered Southern blot patterns after conventional or pulsed-field gel analysis of DNA from APL patients.

Dyskeratosis congenita with linear areas of severe cutaneous involvement.

Dyskeratosis congenita (DC) is a rare hereditary disorder of skin which may be associated with aplastic anemia. The pattern of inheritance is X-linked recessive in most instances, but autosomal dominant and autosomal recessive types have been documented. Reticulated hyperpigmentation usually is the first manifestation. The pigmentary changes may be limited to neck, upper chest, and proximal parts of the limbs initially but within affected areas the involvement is always diffuse. We report on a patient with typical diffuse cutaneous signs of dyskeratosis congenita superimposed with hyperpigmentation that was more pronounced along Blaschko's lines. To explain this phenomenon, we assume that the patient has the autosomal dominant type and that loss of heterozygosity occurred in a somatic cell giving rise to a population of cells that migrated along these lines during embryogenesis.

Toward quality assurance for metaphase FISH: a multi-center experience.

Although fluorescent in situ hybridization (FISH) is rapidly becoming a part of clinical cytogenetics, no organization sponsors multi-center determinations of the efficacy of probes. We report on 23 laboratories that volunteered to provide slides and to use a probe for SNRPN and a control locus. Experiences with FISH for these laboratories during 1994 ranged from 0 to 645 utilizations (median = 84) involving blood, amniotic fluid and bone marrow. In an initial study of hybridization efficiency, the median percentage of metaphases from normal individuals showing two SNRPN and 2 control signals for slides prepared at each site was 97.0 (range = 74-100); for slides prepared by a central laboratory, it was 97.8 (range = 81.6-100). In a subsequent blind study, each laboratory attempted to score 5 metaphases from each of 23 specimens [8 with del(15) (q11.2-->q12) and 15 with normal 15 chromosomes]. Of 529 challenges, the correct SNRPN pattern was found in 5 of 5 metaphases in 457 (86%) and in 4 of 5 in 33 (6%). Ambiguous, incomplete or no results were reported for 32 (6%) challenges. Seven (1%) diagnostic errors were made including 6 false positives and 1 false negative: 1 laboratory made 3 errors, 1 made 2, and 2 made 1 each. Most errors and inconsistencies seemed due to inexperience with FISH. The working time to process and analyze slides singly averaged 49.5 minutes; slides processed in batches of 4 and analyzed singly required 36.9 minutes. We conclude that proficiency testing for FISH using an extensive array of challenges is possible and that multiple centers can collaborate to test probes and to evaluate costs.

Toward quality assurance for metaphase FISH: a multicenter experience.

Although fluorescent in situ hybridization (FISH) is rapidly becoming a part of clinical cytogenetics, no organization sponsors multicenter determinations of the efficacy of probes. We report on 23 laboratories that volunteered to provide slides and to use a probe for small nuclear ribonucleoprotein polypeptide N (SNRPN) and a control locus. Experiences with FISH for these laboratories during 1994 ranged from 0 to 645 utilizations (median = 84) involving blood, amniotic fluid, and bone marrow. In an initial study of hybridization efficiency, the median percentage of metaphases from normal individuals showing two SNRPN and two control signals for slides prepared at each site was 97.0 (range = 74-100); for slides prepared by a central laboratory, it was 97.8 (range = 81.6-100). In a subsequent blind study, each laboratory attempted to score 5 metaphases from each of 23 specimens [8 with del(15)(q11.2-->q12) and 15 with normal #15 chromosomes]. Of 529 challenges, the correct SNRPN pattern was found in 5 of 5 metaphases in 457 (86%) and in 4 of 5 in 33 (6%). Ambiguous, incomplete, or no results were reported for 32 (6%) challenges. Seven (1%) diagnostic errors were made, including 6 false positives and 1 false negative: 1 laboratory made 3 errors, 1 made 2, and 2 made 1 each. Most errors and inconsistencies seemed due to inexperience with FISH. The working time to process and analyze slides singly averaged 49.5 min; slides processed in batches of 4 and analyzed singly required 36.9 min. We conclude that proficiency testing for FISH by using an extensive array of challenges is possible and that multiple centers can collaborate to test probes and to evaluate costs.

A multicenter investigation with D-FISH BCR/ABL1 probes.

Twenty-eight laboratories evaluated a new fluorescence in situ hybridization (FISH) strategy for chronic myeloid leukemia. In a three-part study, bcr/abl1 D-FISH probes were used to study bone marrow specimens. First, laboratories familiarized themselves with the strategy by applying it to known normal and abnormal specimens. Then, collectively the laboratories studied 20 normal and 20 abnormal specimens blindly and measured workload. Finally, each laboratory and two experts studied six serial dilutions with 98-0% abnormal nuclei. Using the reported normal cutoff of < 1% abnormal nuclei, participants reported no false-negative cases and 15 false-positive cases (1-6.6% abnormal nuclei). Results provided by participants for serial dilutions approximated the expected percentages of abnormal nuclei, but those from the experts exhibited greater precision. The clinical sensitivity, precision, nomenclature, workload, recommendations for training, and quality assurance in methods using D-FISH in clinical practice are discussed.

Cytogenetic comparison and phylogeny of three species of Hylobatidae.

Representatives of three subgenera of Hylobatidae, Hylobates (Symphalangus) syndactylus, H. (Nomascus) concolor and H. (Hylobates) agilis were compared karyotypically by G-banding, and silver staining. A greater degree of similarity (30-55%) was found among these groups than previous reports suggest; however, these figures are still considerably lower than chromosome similarities characteristic of all other catarrhine groups. Inversion, translocation, fission, and fusion have all played a role in restructuring hylobatid chromosomes since a common hominoid ancestor. H. syndactylus and H. concolor show the greatest G-band correspondence, and in addition share an unusual C-band distribution and an extremely rare nucleolar organizing region placement (on the Y chromosome). The latter two are probably shared derived traits, suggesting that these two species shared a common ancestor not shared by other hylobatids. These data suggest a branching order for these three hylobatid groups different from those derived by other morphological and biochemical methods.

Heterochromatin, synaptonemal complex, and NOR activity in the somatic and germ cells of a male domestic dog, Canis familiaris (Mammalia, Canidae).

C-banding and silver staining of the somatic and germ cells of the male domestic dog. Canis familiaris, have shown that: (1) the amount of C-banding is small compared to most other mammalian species, (2) three pairs of autosomes have nucleolus organizer regions (NORs) at the terminal ends of their long arms, whereas the Y chromosome has an NOR on the terminal end of the short arm, (3) the organization of the synaptonemal complex (SC) is similar to that of other mammalian species, (4) a distinct SC is formed between the long arm of the Y chromosome and probably the short arm of the X chromosome, and (5) the differential axes of both sex chromosomes do not demonstrate fusiform thickenings nor do they stain darkly with silver as do the XY bivalents in many other mammalian species.

Reduced NOR association frequency in a 13/18 translocation chromosome. A family study.

The association frequency of a functional, intercalary NOR in fibroblasts of five carriers of a translocation t(13;18) was significantly lower than the average association frequency of normal G- and D-group chromosomes in the same carriers. This decreased coefficient of association does not seem to be attributable to partial loss of the NOR during rearrangement or to a loss of NOR activity, as indicated by silver staining. It is hypothesized that either (1) interphase associations including this chromosome form less frequently or, more likely, (2) associations with this chromosome are differentially susceptible to physical disruption due to flattening of association complexes during cell preparation. The presence of this abnormal NOR chromosome did not affect the association behavior of normal G- and D-group chromosomes in carrier cells. In addition, the present study supports previous observations that G-group chromosomes associate with a greater average frequency than D-group chromosomes.

Human liver fatty acid binding protein gene is located on chromosome 2.

The human gene for liver fatty acid binding protein was assigned to chromosome 2 using human-rodent somatic cell hybrids and Southern filter hybridization of cell hybrid DNA. The filters were hybridized to a radiolabeled cloned cDNA which covers 485 bases. Hybridization of the DNA to a human-specific 7.5-kb DNA fragment in HindIII-digested DNA was used to identify the human liver fatty acid binding protein gene. Results from two cell hybrids with chromosomal rearrangements suggest that the gene is on the short arm of chromosome 2.

Localization of the human thyroxine-binding globulin gene to the long arm of the X chromosome (Xq21-22).

Thyroxine-binding globulin (TBG) is the major thyroid-hormone transport protein in the plasma of most vertebrate species. A recombinant phage (lambda cTBG8) containing a cDNA insert of human TBG recently has been described. With the cDNA insert from lambda cTBG8 used as a radiolabeled probe, DNA from a series of somatic-cell hybrids containing deletions of the X chromosome was analyzed by means of blot hybridization. The results indicated that the TBG gene is located in the midportion of the long arm of the X chromosome between bands Xq11 and Xq23. The gene then was mapped to band region Xq21-22 by means of in situ hybridization to metaphase chromosomes. Sequences on the X chromosome that are homologous to the cDNA probe are contained within a single EcoRI restriction fragment of 12.5 kb in human DNA. On the basis of the intensity of the hybridization signal on Southern blots, it was determined that the human TBG cDNA probe used in the present study shares significant homology with hamster and mouse sequences. A single EcoRI restriction fragment was recognized in both hamster (8.0-kb) and mouse (5.1-kb) DNA.

Regional mapping panel for human chromosome 17: application to neurofibromatosis type 1.

A somatic cell hybrid mapping panel was constructed to localize cloned DNA sequences to any of 15 potentially different regions of human chromosome 17. Relatively high-resolution mapping is possible for 50% of the chromosome length in which 12 breakpoints are distributed over approximately 45 megabases, with an average spacing estimated at 1 breakpoint every 2-7 megabases. This high-resolution capability includes the pericentromeric region of 17 to which von Recklinghausen neurofibromatosis (NF1) has recently been mapped. Using 20 cloned genes and anonymous probes, we have tested the expected order and location of panel breakpoints and confirmed, refined, or corrected the regional assignment of several cloned genes and anonymous probes. Four markers with varying degrees of linkage to NF1 have been physically localized and ordered by the panel: the loosely linked markers myosin heavy chain 2 (25 cM) to p12----13.105 and nerve growth factor receptor (14 cM) to q21.1----q23; the more closely linked pABL10-41 (D17S71, 5 cM) to p11.2; and the tightly linked pHHH202 (D17S33) to q11.2-q12. Thus, physical mapping of linked markers confirms a pericentromeric location of NF1 and, along with other data, suggests the most likely localization is proximal 17q.

A study of chromosomal aberrations and chromosomal fragility after recombinant growth hormone treatment.

Increased chromosomal rearrangements and chromosomal fragility have been previously observed in lymphocytes of children treated with human GH, implying that treatment could predispose to malignancy. Twenty-four children with classic GH deficiency, neurosecretory GH dysfunction, and Turner syndrome were treated with recombinant human GH (0.3 mg x kg(-1) x wk(-1)). Metaphase cells were assessed for spontaneous chromosomal and chromatid aberrations at baseline and 6 mo into treatment. There were no significant differences in aberrations between baseline and the 6-mo samples. However, the mean frequency of chromatid-type aberrations on a per cell basis was significantly higher than at baseline, 0.0088 versus 0.0064 aberrations per cell (p < 0.024). Two patients contributed inordinately to this increase. A third sample from these two patients was almost identical to their baseline samples. Cells were also irradiated in vitro (3 Gy) to assess chromosomal fragility. After irradiation, no patient showed a significant difference for any aberration type, although there was a significantly lower frequency of ring chromosomes on a per cell basis in the 6-mo samples (p < 0.001). We find no evidence that GH therapy influences spontaneous chromosomal aberrations or chromosomal fragility.

Complex, compound inversion/translocation polymorphism in an ape: presumptive intermediate stage in the karyotypic evolution of the agile gibbon Hylobates agilis.

Karyotypic variation in five gibbon species of the subgenus Hylobates (2n = 44) was assessed in 63 animals, 23 of them wild born. Acquisition of key specimens of Hylobates agilis (agile gibbon), whose karyotype had been problematic due to unresolved structural polymorphisms, led to disclosure of a compound inversion/translocation polymorphism. A polymorphic region of chromosome 8 harboring two pericentric inversions, one nested within the other, was in turn bissected by one breakpoint of a reciprocal translocation. In double-inversion + translocation heterozygotes, the theoretical meiotic pairing configuration is a double inversion loop, with four arms of a translocation quadrivalent radiating from the loop. Electron-microscopic analysis of synaptonemal complex configurations consistently revealed translocation quadrivalents but no inversion loops. Rather, nonhomologous pairing was evident in the inverted region, a condition that should preclude crossing over and the subsequent production of duplication-deficiency gametes. This is corroborated by the existence of normal offspring of compound heterozygotes, indicating that fertility may not be reduced despite the topological complexity of this polymorphic system. The distribution of inversion and translocation morphs in these taxa suggests application of cytogenetics in identifying gibbon specimens and avoiding undesirable hybridization in captive breeding efforts.

Isolation and characterization of radiation-reduced hybrids containing portions of the proximal long arm of the human X chromosome: identification of hybrids containing the Menkes' disease locus.

The proximal long arm of the human X chromosome (Xcen----Xq13) encompasses an estimated 23 megabases of DNA and contains numerous identified genetic loci. In order to generate a highly enriched source of DNA from this region, radiation-reduced human-hamster hybrids were constructed and screened to identify those that contained at least part of proximal Xq. Eight such hybrids were identified and characterized by Southern blot and fluorescence in situ hybridization analyses to determine more precisely the human DNA complement in each. One hybrid contains the entire proximal long arm and will be useful for mapping Xcen----Xq13 in its entirety and for localizing genes within this region. Another hybrid contains a smaller portion of the proximal long arm that includes the region reported to contain the gene for Menkes' disease.

Assignment of human tryptophan hydroxylase locus to chromosome 11: gene duplication and translocation in evolution of aromatic amino acid hydroxylases.

A cDNA clone for rabbit tryptophan hydroxylase was used as a probe to identify human tryptophan hydroxylase gene fragments in a panel of hamster-human somatic cell hybrids and determine its chromosomal location in man. A single locus was identified for tryptophan hydroxylase on chromosome 11. Tryptophan hydroxylase is a member of the superfamily of pterin-dependent aromatic amino acid hydroxylases which includes tyrosine hydroxylase, located at 11p15.5-p15, and phenylalanine hydroxylase, located at 12q22-q24.1 in human. The locations of these genes and the evolutionary distance between their sequences suggest that at least three distinct genetic events have occurred during the evolution of the aromatic amino acid hydroxylase superfamily: two sequential gene duplications giving rise to the three distinct hydroxylase loci, and a translocation which separated the tryptophan and tyrosine hydroxylase loci on chromosome 11 from the phenylalanine hydroxylase locus on chromosome 12.

A myeloid-related sequence that localizes to human chromosome 8q21.1-22.

A myeloid-related sequence (mrs) has previously been identified that is highly expressed in selected subpopulations of myeloid leukocytes. Nucleotide sequence analysis indicates that mrs encodes what is apparently a unique 93-amino acid protein that includes an 18-amino acid leader sequence. Hybridization of an mrs cDNA probe to a Southern blot made from somatic cell hybrid DNAs shows 100% concordance with human chromosome 8, thus indicating that mrs localizes to this chromosome. In situ hybridization to metaphase chromosomes further sublocalizes mrs to bands 8q21.1-23 as 58% of the grains displayed on chromosome 8 were clustered in this region. This area encompasses the translocation breakpoint 8q22, which is rearranged in an estimated 18% of patients diagnosed with the M2 subclassification of acute nonlymphocytic leukemia (M2-ANLL). When Southern blot hybridization was performed by using somatic cell hybrid DNAs harboring either a single 8q- or a single 21q+ chromosome from two different patients with M2-ANLL, a signal was only detected in the hybrid containing the 8q-chromosome.

Construction of a YAC contig encompassing the Usher syndrome type 1C and familial hyperinsulinism loci on chromosome 11p14-15.1.

The Usher syndrome type 1C (USH1C) and familial hyperinsulinism (HI) loci have been assigned to chromosome 11p14-15.1, within the interval D11S419-D11S1310. We have constructed a yeast artificial chromosome (YAC) contig, extending from D11S926 to D11S899, which encompasses the critical regions for both USH1C and HI and spans an estimated genetic distance of approximately 4 cM. A minimal set of six YAC clones constitute the contig, with another 22 YACs confirming the order of sequence-tagged sites (STSs) and position of YACs on the contig. A total of 40 STSs, including 10 new STSs generated from YAC insert-end sequences and inter-Alu PCR products, were used to order the clones within the contig. This physical map provides a resource for identification of gene transcripts associated with USH1C, HI, and other genetic disorders that map to the D11S926-D11S899 interval.

Human interstitial retinol-binding protein (IRBP): cloning, partial sequence, and chromosomal localization.

A cloned 2184-bp cDNA coding for human interstitial retinol-binding protein (IRBP) has been isolated and sequenced. The probe hybridized to a 5.2-kb poly(A) RNA from human retinas. Nineteen tryptic peptides (363 amino acids) sequenced and purified from bovine IRBP could be aligned with 86-88% homology to the translated sequence. Two segments approximately 200 amino acids long were found to have a 41% residue identity, suggesting an internal duplication event. This cloned cDNA was used to probe DNA samples from a panel of 29 rodent-human somatic cell hybrids, mapping the structural gene for IRBP to chromosome 10. In situ hybridization suggested a regional localization near the centromere (p11.2----q11.2), although a secondary site of hybridization at q24----25 was also observed.

Alteration and abnormal expression of the c-myc oncogene in human multiple myeloma.

Structural alterations of the c-myc oncogene in human Burkitt's lymphoma and mouse plasmacytoma suggest that this oncogene is involved in several B cell neoplasms. The possibility of c-myc alterations in human myeloma has not been explored, probably because the low proliferative activity characteristic of this tumor impairs the propagation of representative cell lines for the performance of adequate cytogenetic studies. This report describes alterations in the c-myc locus with concomitant elevated expression of mRNA in the tumor cells of two of 37 patients with multiple myeloma. In one case, somatic cell hybrid studies revealed that the cloned rearranged DNA was entirely derived from chromosome 8, thus indicating a novel mechanism of c-myc activation different from that in Burkitt's lymphoma. Seven other patients exhibited five- to 12-fold overexpression of c-myc RNA when compared with normal marrow cells. Elevated mRNA expression in about one fourth of our patients suggests that the c-myc oncogene has a pathogenetic role in the evolution of multiple myeloma.