Partial dystrophin deficiency in monozygous twin carriers of the Duchenne gene discordant for clinical myopathy.

We studied monozygous twin women, age 63. One, asymptomatic, had a serum creatine kinase (CK) level of 191 units (normal, 1 to 50); her son died of typical Duchenne muscular dystrophy (DMD) at age 18. Her twin sister had symptomatic limb weakness from about age 40. Her serum CK was 495 units. EMG and muscle biopsy were compatible with myopathy. In the asymptomatic twin, the peripheral blood lymphocyte karyotype was 46,XX. In the affected twin, 18% of cells were 45,X, and the others 46,XX, without X/autosome translocation. DNA analysis did not reveal a deletion at the DMD locus. Immunologic studies of dystrophin showed a partial deficiency of the protein that was more severe in the symptomatic twin. The clinical discordance and the different severity of dystrophin deficiency may have resulted from the effects of lyonization.

Inhibins and activins in human fetal abnormalities.

To date, the only routine clinical application of inhibin or activin measurement in testing for fetal abnormalities has been the use of inhibin A in prenatal screening for trisomy 21 (Down syndrome). Second trimester maternal serum levels of inhibin A are, on average, two-fold higher in Down syndrome than in unaffected pregnancies. Although the biology of altered second trimester maternal serum analyte levels in Down syndrome pregnancy cannot yet be explained, it seems that fetal products tend to be decreased, while placental products tend to be increased. This pattern holds true for inhibin A because maternal serum levels appear to be derived from placental rather than fetal sources. Therefore, the measurement of inhibins and activins in maternal fluids, although clinically useful and relatively easy to obtain, may not be helpful in studying their role in human fetal development. Studies in transgenic mice indicate a role for activin, follistatin, and activin receptor type IIA in development of the palate and craniofacial region. Cleft palate is a common birth defect and is associated with serious feeding and respiratory complications in newborns. We have begun to investigate the potential role of activin in human craniofacial development by examining the spatial and temporal expression of inhibin/activin subunits, follistatin and the activin receptors in the fetal palate. Palate tissues were collected at autopsy from fetuses ranging in gestational age from 9 to 42 weeks, and 8 week embryonic tissues were also examined. Tissues were either stored in paraffin for immunocytochemistry or were frozen for RT-PCR examination of the expression of inhibin/activin proteins or mRNAs, respectively. To date, betaA subunit, follistatin, and activin receptor, but not alpha and betaB subunit, mRNAs are present in palate tissues and inhibin/activin betaA immunoreactivity has been consistently observed in developing bone. Expression of the activin A subunit and its receptors in the human fetal palate are consistent with a developmental role. Studies are ongoing to determine whether altered activin biosynthesis is associated with cleft palate. Future studies of fetal tissues may help to elucidate other roles for the TGF-beta family in human development.

Quantitative calibration and use of DNA probes for investigating chromosome abnormalities in the Prader-Willi syndrome.

Ten genomic DNA probes, subcloned from inserts derived from a phage library constructed from the DNA of flow-sorted chromosomes, have now been mapped to locations within 15q11-15q13. By dosage blotting and densitometry, 5 of these probes map to the 15q11.2-15q12 segment missing in one 15 chromosome of a Prader-Willi syndrome (PWS) patient with a prominent cytological deletion. A sixth probe most likely maps to the same region. The other 4 probes map outside of this segment but within 15q11-15q13. Several of the 15q11.2-15q12 probes, and a cDNA probe homologous to one, have been used to test the DNA from 8 patients exhibiting a wide range of the clinical manifestations expected for PWS patients. DNA deletion was observed in all 3 patients with cytological 15q1 deletions as well as in a patient with an unbalanced (Y;15) translocation. DNA from 1 PWS patient with an unbalanced (5;15) translocation and an inverted duplication of the short arm and proximal long arm of 15 showed at least 1 and possibly 2 extra copies of each genomic probe tested. In the other 3 patients with no cytological deletions, no DNA deletions were found. Thus, the molecular probes described can be used in most PWS patients to analyze the region of proximal 15q implicated in this syndrome.

5alpha-reductase 1 and 2 expression and activity in human ovarian follicles, stroma and corpus luteum as compared to neonatal foreskin.

5alpha-Reductase is the steroidogenic enzyme which reduces testosterone to 5alpha-dihydrotestosterone. In the human two different enzymes have been described, 5alpha-reductase 1 and 2. The present investigations were undertaken to determine whether 5alpha-reductase 1 and 2 were expressed in the human ovary, and to determine the relative activity of the two enzymes in various ovarian tissues. The ovary apparently expressed mRNA for only 5alpha-reductase 1, whereas the foreskin expressed both 5alpha-reductase 1 and 2. We compared the 5alpha-reductase activity at both pH 5.5 (optimum for 5alpha-reductase 2 activity) and 8.0 (optimum for 5alpha-reductase 1 activity). 5alpha-reductase activity of foreskin at pH 5.5 was 3900 times higher than small follicles, 1500 times higher than ovarian stroma, and 240 times higher than corpora lutea (all P < 0.01). 5alpha-reductase activity of corpora lutea at pH 5.5 was 17-fold higher than that of follicles (P < 0.01) and 6.5-fold higher than that of ovarian stroma (P < 0.05). 5alpha-Reductase activity of foreskin at pH 8.0 was 93 times higher than small follicles, 51 times higher than corpora lutea, and 170 times higher than ovarian stroma (all P < 0.01). The ratio of 5alpha-reductase activity at pH 5.5 to that at pH 8.0 was higher in foreskin than in corpus luteum (P < 0.05), ovarian stroma (P < 0.01), or ovarian follicles (P < 0.01). The ratio was lower in ovarian follicles than in stroma or corpus luteum (both P < 0.05).

The rat XC sarcoma cell line: ribosomal RNA gene amplification and banded karyotype.

Ribosomal RNA gene amplification has been demonstrated in the rat XC sarcoma cell line. Cells of this rat line have a fairly stable karyotype with several unusual features, which have been clarified by in situ hybridization, silver staining, and binding of antibodies to 5-methylcytosine. There are one or two tiny acrocentric chromosomes containing transcriptionally active 18S and 28S ribosomal RNA (rRNA) genes. The short arm of one chromosome No. 12 has been replaced by a GC-rich, C-band negative, differentially staining region (DSR) containing an increased number of rRNA genes; most of these are transcriptionally inactive and located in regions containing highly methylated DNA. The short are of a small chromosome, probably a No. 20, has been replaced by a GC-rich, C-band negative, homogeneously staining region (HSR) that presumably represents amplification of a DNA sequence other than the 18S and 28S rRNA coding sequences. The DNA in this HSR is not enriched in 5-methylcytosine and neither is that in the HSRs of methotrexate-resistant Syrian hamster cells.

Homology of lubricin and superficial zone protein (SZP): products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25.

We have previously identified megakaryocyte stimulating factor (MSF) gene expression by synovial fibroblasts as the origin of lubricin in the synovial cavity. Lubricin is a mucinous glycoprotein responsible for the boundary lubrication of articular cartilage. MSF has a significant homology to vitronectin and is composed of 12 exons. RNA was purified from human synovial fibroblasts and articular chondrocytes grown in vitro from tissue explants obtained from subjects without degenerative joint disease. RT-PCR was used with multiple complimentary primer pairs spanning the central mucin expressing exon 6 of the MSF gene and individual exons on both the N- and C-terminal sides of exon 6. Exons 2, 4 and 5 appear to be variably expressed by synovial fibroblasts and articular chondrocytes. Lubricating mucin, in the form of MSF, is expressed by both chondrocytes and synovial fibroblasts in vitro. Both lubricin and superficial zone protein (SZP), a related proteoglycan, share a similar primary structure but could differ in post-translational modifications with O-linked oligosaccharides which are predominant in lubricin and with limited amounts chondroitin and keratan sulfate found in SZP. Since most of the MSF exons are involved in the expression of lubricating mucin, a strong homology to vitronectin persists. It is therefore appropriate to consider that both SZP and lubricin occupy a new class of biomolecules termed tribonectins. Screening of a human genome bacterial artificial chromsome (BAC) library with a cDNA primer pair complimentary for exon 6 identified two clones. Both clones were complimentary for chromosome 1q25 by in situ hybridization. This same locus was previously implicated in camptodactyl-arthropathy-pericarditis syndrome (CAP) by genetic mapping. It is hypothesized that CAP, a large joint arthropathy, may be associated with ineffective boundary lubrication provided by synovial fluid.

Correspondence between effects of 5-azacytidine on SCE formation, cell cycling and DNA methylation in Chinese hamster cells.

The effects of 5-azacytidine (5-Aza-C), alone and in combination with mitomycin C, were measured on sister-chromatid exchange (SCE) formation and DNA methylation in different genomic regions of Chinese hamster ovary cells and in Chinese hamster cells containing amplified, dihydrofolate reductase sequences and resistant to methotrexate. 5-Aza-C, when present for the penultimate preharvest cell cycle, induced SCEs in a manner consistent with a directly measured reduction in deoxycytosine methylation in cellular DNA. At higher 5-Aza-C concentrations, cell cycling was inhibited and both SCE induction and DNA demethylation tended to level off. Under appropriate conditions, 5-Aza-C also potentiated the induction of SCEs by mitomycin C. 5-Aza-C-induced DNA demethylation could also be detected in the vicinity of different DNA sequences with the use of comparative HpaII/MspI digestion, DNA blotting, and molecular probes. The efficiency of an individual demethylation event in inducing SCE induction appeared to be very low, compared with alkylating agents such as 8-methoxypsoralen, suggesting that SCE induction by 5-Aza-C might be an indirect effect from long range changes induced in cellular DNA or chromatin conformation.

Biological effects of a bifunctional DNA cross-linker. II. Generation of micronuclei and attached micronuclear-like structures.

Madin-Darby bovine kidney (MDBK) cells were treated with the bifunctional DNA cross-linker, L-7, to examine the generation of micronuclei and other nuclear abnormalities. The preceding paper demonstrates that L-7 treatment induces the formation of triradial and quadriradial chromosomes in MDBK cells. These chromosomes are believed to result from interduplex DNA cross-links formed between G-C rich centromeric satellite DNA regions on non-sister chromatids. Treatment produces a majority of centromere-positive micronuclei. In addition, many daughter cells remain attached by chromatin bridges which are sometimes beaded with micronuclei. Up to 15% of cell nuclei become lobular and fused with numerous micronuclear-like structures attached to their membranes. These attached structures are classified as attached micronuclear-like structures (AMNLS). Fluorescence in situ hybridization (FISH) using a centromeric satellite sequence was performed on treated cells. Hybridization reveals that intercellular bridges are composed of centromeric sequences and initiate at centromeric foci in daughter cells. Furthermore, the majority of junctions between AMNLS and nuclei contain an enhancement of centromeric signal. The frequency of AMNLS appears dependent on the concentration of L-7 and the duration of treatment. Similar results were found for the generation of cross-linked chromosome products in the previous paper. We suggest that AMNLS result from the abnormal mitotic segregation of cross-linked chromosome products.

Biological effects of a bifunctional DNA crosslinker. I. Generation of triradial and quadriradial chromosomes.

Interduplex crosslinks by a bifunctional anthramycin DNA crosslinker produced triradial and quadriradial chromosomes. The crosslinker alkylates guanine at N-2. Bovine chromosomes contain GC-rich density satellite DNAs at the centromeric heterochromatin and is the basis for the formation of triradial and quadriradial chromosomes at the centromeres. The in situ crosslinking of interphase chromosomes indicates that the distance between centromeres is 17.5 A. We conclude that the nuclear matrix associated DNA in the centromeric heterochromatin of interphase chromosomes are positioned close enough for crosslinking to occur. We propose a model for the generation of triradial and quadriradial chromosomes based upon the number of interduplex crosslinks between two chromosomes.

Overexpression of the HER-2/neu oncogene in pancreatic adenocarcinoma.

Novel systemic treatments are needed in pancreatic cancer. The authors sought to establish the frequency of overexpression of the HER-2/neu oncogene in patients with pancreatic adenocarcinoma to determine the potential role of trastuzumab (Herceptin) as a therapeutic agent in this disease. Tumor specimens from patients with pancreatic adenocarcinoma were analyzed by staining for p185HER2 protein using the DAKO immunohistochemical assay. Patients with and without HER-2/neu overexpression by immunohistochemistry were compared with respect to clinical and pathologic characteristics. HER-2/neu gene amplification was also evaluated by fluorescence in situ hybridization (FISH). Thirty-two of 154 patients (21%) had pancreatic adenocarcinoma that demonstrated HER-2/neu overexpression by immunohistochemistry. At initial diagnosis, 16% of resectable cancers, 17% of locally advanced cancers, and 26% of metastatic cancers were determined to have HER-2/neu overexpression. Three of 11 (27%) patients with HER-2/neu overexpression by immunohistochemistry had gene amplification by FISH. HER-2/neu overexpression occurs in a subset of pancreatic cancer. Evaluation of the efficacy of trastuzumab for patients with pancreatic cancer who overexpress HER-2/neu appears indicated.

Bilateral semilunar valve dysplasia in a patient with inverted duplication 2p25-22.

Here we report a patient with partial trisomy 2p and congenital dysplasia of the semilunar valves. To our knowledge, this is the first case of 2p duplication with developmental defects of both semilunar valves and suggests that genes on this region contribute to the formation of the semilunar valves.

46,XX gonadal agenesis in a neonate with multiple congenital anomalies: case report and review of the literature.

We report a neonate with 46,XX gonadal agenesis, a rare disorder, confirmed by autopsy, karyotype determination, and fluorescent in situ hybridization examination of intact cells. Multiple other anomalies, including diaphragmatic hernia, a doomed bicuspid aortic valve, and müllerian derivative defects, were present. There was no sexual ambiguity. The age of this patient and the presence of anatomically dispersed congenital anomalies are unique among reported examples of 46,XX gonadal agenesis. Review of the literature reveals that all five previously reported cytogenetically confirmed patients with 46,XX gonadal agenesis were 17 to 25 years of age, none were diagnosed before their teens, all had female phenotype with sexual infantilism, three had müllerian derivative anomalies, and none had nongenitourinary anomalies. The abnormalities in this case may represent a polytopic field defect due to unknown insults occurring at approximately 6 weeks of developmental age.

Immunocytochemical evidence for methylation of the inactive X chromosome in human fetal oogonia.

The state of DNA methylation of the X chromosomes of human interphase oogonia from a 46,XX and a 46,XX/47,XXX fetus at 17 weeks of gestation was tested immunocytochemically with an antibody to 5-methylcytosine (5MeC). Of 1637 oogonial nuclei from the 46,XX fetal ovary, 313 (19.1%) contained Barr bodies, of which 93.6% were positive for 5MeC. Of 1780 oogonia from the 46,XX/47,XXX fetus 327 (18.4%) contained Barr bodies; 175 oogonia had one Barr body and 152 had two. Of the single Barr bodies 145 (82.8%) had positive 5MeC reaction product. Of the 152 oogonia from the XXX line, 97 (63.8%) had positive 5MeC on both Barr bodies, 35 (23%) had one positive and one negative, and 20 (13.1%) had no product on either Barr body. This immunocytochemical evidence supports the hypothesis that the DNA of the inactive X-chromosome of the human 17-week gestation oogonium is methylated.

Parental imprinting of an Igf-2 transgene.

As a consequence of parental imprinting in mice, the paternal allele encoding insulin-like growth factor-II (IGF-II) is expressed, whereas the maternal allele is silent in most tissues. To examine whether cis-acting sequences involved in imprinting are located in the vicinity of the Igf-2 gene, we have constructed mouse transgenic lines and studied the expression of a 30 kb rat Igf-2 transgene, in which the coding region has been replaced with the lacZ reporter sequence. Chromatin position effects and/or absence of long-range regulatory elements seem to have affected tissue-specific expression in the transgenic mice. However, in one of six expressing lines, staining of embryos for beta-galactosidase activity was detected in a minor subset of tissues normally transcribing the endogenous homolog, but only when the transgene was transmitted paternally. This transgene was integrated into mouse chromosome 19, which is apparently free of imprinted loci. Although the possibility that the Igf-2 transgene was inserted into an as yet unidentified imprinted locus is discussed, a more likely interpretation of our results is that the transgene carries at least a portion of its own imprinting signal, because it consists of the genomic sequences of a locus already known to be imprinted and maintains the correct imprinting mode.

Trisomy 16 mosaicism in amniotic fluid cell cultures.

Trisomy 16 mosaicism was found in amniotic fluid cells in a patient undergoing amniocentesis because of elevated second-trimester maternal serum alpha-fetoprotein (MSAFP) (2.80 MOM), a markedly elevated human chorionic gonadotropin level (hCG) (12.02 MOM), and a Down syndrome risk of 1:55. Ultrasound evaluation of the fetus indicated the presence of an atrial septal defect and clinodactyly. Cytogenetic analyses of various fetal tissues using fluorescence in situ hybridization (FISH) failed to detect substantial numbers of trisomy 16 cells; however, trisomy 16 mosaicism was identified in placental tissue. Molecular genetic analysis at five different loci [four analysed by polymerase chain reaction (PCR) and one by Southern blot analysis] failed to show any evidence for uniparental disomy. Although trisomy 16 cells could not be clearly demonstrated in the fetus, the presence of a clinically significant proportion of aneuploid cells early in development could not be excluded and it therefore cannot be assumed that a 'confined placental mosaicism' existed. The markedly elevated hCG and elevated MSAFP levels are consistent with abnormal placental function in trisomy 16 mosaicism. Serial ultrasound evaluation (to detect any late-onset growth retardation) and fetal echocardiography may be indicated for patients with extraordinarily high levels of hCG, especially if MSAFP is also elevated.

Cytologic and molecular analysis of 46,XXq- cells to identify a DNA segment that might serve as a probe for a putative human X chromosome inactivation center.

Cloned human X chromosome-specific DNA segments, derived from a recombinant phage library enriched for the human X and previously localized to different regions of the X, were used as probes in Southern blots to confirm the nature of a deletion of the long arm of the X chromosome as del(X)(q13) in a patient with some features of Turner's syndrome and suspected from cytologic studies to have a 46,XXq- karyotype. Two dimensional scanning densitometry of autoradiograms of the Southern blots was used to quantitate hybridization of the 32P-labeled probes, reinforcing visual analysis and permitting distinction between sequences present at one or two copies per diploid genome. Once thus characterized, DNA from the patient's cells was used in quantitatively analyzed Southern blots to refine the location of an additional DNA segment, previously mapped to somewhere in the proximal part of the long arm of the X chromosome, to the juxtacentromeric region of Xq, which has been hypothesized to be critical for X-inactivation. Cloned DNA probes such as that localized to the juxtacentromeric region of Xq should be useful for evaluating this hypothesis.

Homogeneously staining regions (HSRs) of a rat hepatoma cell line are not early replicating.

The rat hepatoma cell line H4-IIE-C3 (H4) has homogeneously staining regions (HSRs) which contain multiple, tandemly repeated copies of ribosomal RNA (rRNA) genes. We determined the time of replication of the DNA within these HSRs autoradiographically after incorporation of [3H]thymidine and by Hoechst 33258 and Giemsa staining after 5-bromodeoxyuridine (5-BrdU) incorporation. The DNA within the H4 HSRs is not early replicating, unlike that in other HSRs. It begins replicating later than much of the other nuclear DNA, continues replicating throughout most of the S phase, and is completed 1-2 h before mitosis.

Isolation and characterization of two repetitive DNA fragments located near the centromere of the mouse X chromosome.

Two repetitive DNA fragments located on the mouse X chromosome are described. The fragments were isolated from a lambda phage library enriched in X-chromosomal sequences by flow sorting. Both fragments, which are repeated 20 to 50 times in the genome, were mapped to the mouse X chromosome by Southern blot hybridization to DNA from hybrid cells retaining the mouse X chromosome, by dosage analysis, and by in situ hybridization to mouse chromosomes. In mouse strain C57BL/10BK, one fragment appeared to be located only on the X chromosome, while the other fragment had homologous sequences on chromosome 11 in addition to the X chromosome. The latter fragment showed DNA variants between mouse strains, which are potentially useful for mapping. Both fragments cross-hybridized to another mouse species: Mus caroli. In this species, each fragment appeared to be located on the X chromosome, indicating that some X-chromosome repetitive sequences are partially conserved. In addition, one fragment cross-hybridized to human DNA.