20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits.

BACKGROUND: Wolff-Parkinson-White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. METHODS AND RESULTS: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. CONCLUSIONS: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Superradiant emission dynamics of an optically thin material sample in a short-decay-time optical cavity

We report observations of optical superradiant emission and the atomic evolution it drives under conditions closely approximating those originally envisioned in the classic work of Dicke [Phys. Rev. 93, 99 (1954)]. Our experiment involves an optically thin solid sample in a short-lifetime optical cavity whose homogeneous coherence is cryogenically stabilized. Pulsed coherent excitation initiates superradiant emission which subsequently drives the sample to higher or lower states of coherence. Suppression of dephasing via cryogenics and propagation effects through use of an optically thin sample and cavity provides one of the clearest and cleanest examples of Dicke superradiance yet reported.

Immunofluorescent studies of human chromosomes with antibodies against phosphorylated H1 histone.

One of the prominent cell cycle-related modifications of histone proteins whose function remains unresolved is the phosphorylation of linker histone H1. In this work we have used indirect immunofluorescence on human cells with antibodies that are specific for phosphorylated histone H1 to examine the cellular distribution and chromosome association patterns of this protein. With confocal microscopy on whole cells, strong immunofluorescence was seen in association with mitotic chromosomes as well as a prominent punctate pattern of labeling throughout the mitotic cell, whereas interphase cells showed very little, if any, specific fluorescence. Multiple patterns of fluorescence distribution were detected with metaphase chromosomes, ranging from apparent tight colocalization with the DNA to expanded "puffy" mitotic figures to an amorphous network of staining. It was also shown that the ability to label chromosomes could vary drastically with different fixation procedures, adding further complications to interpretation of the potentially complex role of phosphorylated histone H1 in chromatin condensation or decondensation.

Laser frequency stabilization by means of optical self-heterodyne beat-frequency control.

We propose and demonstrate a novel frequency-stabilization scheme that provides linewidth narrowing and at the same time is compatible with high-speed frequency agility. The method relies on sensing and control of a heterodyne beat signal derived from a fiber interferometer and functions in the absence of a fixed reference frequency. Our demonstration utilizes a short-external-cavity diode laser equipped with an intracavity electro-optic crystal for frequency correction. The stabilization method is shown to suppress impressed laser frequency modulation by nearly 2 orders of magnitude. Without further modification of our scheme we also demonstrate laser frequency tuning control.

Simple high-coherence rapidly tunable external-cavity diode laser.

We describe a simple electro-optically activated external-cavity diode laser designed to provide high-speed, high-coherence tuning over gigahertz frequency ranges. Tuning as fast as 23 GHz/ micros is demonstrated. Coherence measurements indicate transform-limited quiescent laser linewidth in observation windows as wide as ~100 micros and transform-limited chirps. A self-heterodyne, cross-correlation-based coherence diagnostic is developed for characterizing phase coherence during high-speed chirps.

Analysis of DNA replication by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) has been shown to discriminate between unreplicated and replicated regions of the genome in interphase nuclei, based on the number of specific fluorescent signals that can be detected. By examining the replication status of hybridizing sequences in large numbers of individual cells from an asynchronously growing population, it is possible to deduce a relative order of replication of different sequences. The availability of well-mapped genomic probes and the ability to compare results from different cell lines make this a convenient approach with which to map domains of replication timing control at any chromosomal position and to relate this to various patterns of gene expression. Since there appear to be important but poorly understood correlations among replication timing, chromatin structure, and transcriptional competence in mammalian cells, this provides a valuable approach to understanding these interrelationships at the molecular level. The procedures for using FISH to examine replication timing in mammalian nuclei are described here in detail, and the advantages and limitations of the approach are discussed. Some other strategies for using high-resolution FISH on chromatin fibers to examine replication properties of specific sequences in situ are also described.

Reduced levels of histone H3 acetylation on the inactive X chromosome in human females.

Novel antibodies were generated that are highly selective for either acetylated or unacetylated isoforms of histone H3, or the acetylated form of histone H4 in organisms as diverse as Tetrahymena and humans. Using these antibodies as pair-wise sets in immunocytological analyses, we demonstrate that the inactive X chromosome is hypoacetylated for both histone H3 and H4 in female mammalian cells, whereas the antibody that recognizes the unacetylated form of histone H3 identifies all chromosomes uniformly. These data verify and extend previous results and suggest that hypoacetylation of core histones may be a general feature of the chromatin along the inactive X chromosome.

Significant divergence in nucleotide sequences for beta-tubulin from different laboratory strains of Chinese hamster ovary cells.

The nucleotide sequences for isotype 1 beta-tubulin cDNAs cloned from different laboratory strains of Chinese hamster ovary (CHO) cells were compared and found to contain an unexpected number of sequence differences in both translated and untranslated regions of the gene. The results indicate significant changes in the DNA, but not protein, sequence while the cells have been in culture and reveal sequences in the 5' and 3' untranslated regions that have resisted these changes.

Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization on interphase nuclei of normal female cells to compare the replication timing patterns of genes on the human X chromosome that are known to escape X inactivation with those that are inactivated. By this procedure it was possible not only to determine the relative time of replication of the earlier-replicating allele for different loci but also to estimate the degree of asynchrony of replication of the two alleles for each individual locus. Loci such as HPRT and FRAXA, which are normally inactivated, displayed a high degree of replication asynchrony, whereas loci that are not inactivated (ZFX and RPS4X) were found to replicate very synchronously. Interestingly, examination of XIST, which is expressed only from the inactive X chromosome, by this procedure revealed that it also replicated asynchronously, with the expressed copy apparently replicating first. Therefore, by examining different loci from the X chromosome it was determined that there is a strict correlation between the expression and relative time of replication of individual genes.

Identification of methionine-containing tryptic peptides of unstable beta-tubulin separated by reverse-phase high-performance liquid chromatography.

Methods for examining altered regions in unstable mutant proteins are described. The strategy is illustrated using assembly defective Chinese hamster beta-tubulin subunits that are rapidly degraded in the cell. These unstable proteins are metabolically labeled to high specific activity and isolated as spots on two-dimensional gels. Conditions for the generation of tryptic peptides from gel pieces containing beta-tubulin and their subsequent resolution by HPLC have been worked out. Through a combination of dual labeling with various tritiated amino acids and [35S]methionine as well as partial sequence analysis, the identification of several HPLC peaks with the known sequence of beta-tubulin has been accomplished. This technique should greatly aid attempts to map the sites of mutational alterations in beta-tubulin polypeptides, and the general strategy should be readily applicable to other mutant proteins.

Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus.

The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line. Localization of the missing sequence in D2 was first determined by tryptic peptide mapping by high performance liquid chromatography. Subsequently, the assignment was confirmed by constructing appropriate subclones of a wild type Chinese hamster ovary beta-tubulin cDNA for Southern analysis to demonstrate a failure to recognize characteristic hybridization patterns of the mutant tubulin gene. In the other revertant, 6H3, the deletion was detected on a Northern blot by differential hybridization of a 3' fragment of the cDNA to the beta-tubulin messages. The results indicate that D2 has an internal deletion whose approximate limits extend from amino acid residues 250 through 345. Cell line 6H3 has a deletion that begins near amino acid residue 330 and extends into the 3'-untranslated region of the gene.

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

Optimal myocardial preservation with an acalcemic crystalloid cardioplegic solution.

The effect of the calcium and oxygen contents of a hyperkalemic glucose-containing cardioplegic solution on myocardial preservation was examined in the isolated working rat heart. The cardioplegic solution was delivered at 4 degrees C every 15 minutes during 2 hours of arrest, maintaining a myocardial temperature of 8 degrees +/- 2 degrees C. Hearts were reperfused in the Langendorff mode for 15 minutes and then resumed the working mode for a further 30 minutes. Groups of hearts were given the oxygenated cardioplegic solution containing an ionized calcium concentration of 0, 0.25, 0.75, or 1.25 mmol/L or the same solution nitrogenated to reduce the oxygen content and containing 0 or 0.75 mmol ionized calcium per liter. The myocardial adenosine triphosphate concentrations at the end of arrest in these six groups of hearts were 15.6 +/- 1.2, 9.5 +/- 0.5, 8.2 +/- 1.1, 4.9 +/- 1.8, 10.1 +/- 2.0, and 1.6 +/- 0.4 nmol/mg dry weight, respectively. At 5 minutes of working reperfusion, the percentages of prearrest aortic flow were 80 +/- 2, 62 +/- 4, 33 +/- 6, 37 +/- 5, 48 +/- 7 and 46 +/- 8, respectively. The differences among the groups in adenosine triphosphate concentrations and in functional recovery diminished during reperfusion. In hearts given the hypoxic calcium-containing solution, there was a marked increase in coronary vascular resistance during the administration of successive doses of cardioplegic solution, which was rapidly reversible upon reperfusion. These data indicate that hearts given the acalcemic oxygenated solution had better adenosine triphosphate preservation during arrest and better functional recovery than hearts in any other group. Addition of calcium to the oxygenated cardioplegic solution decreased adenosine triphosphate preservation and functional recovery. Oxygenation of the acalcemic solution increased adenosine triphosphate preservation and functional recovery. The lowest adenosine triphosphate levels at end arrest were observed in hearts given the hypoxic calcium-containing solution. In the setting of hypothermia and multidose administration, the addition of calcium to a cardioplegic solution resulted in increased energy depletion during arrest and depressed recovery.

The superiority of cold oxygenated dilute blood cardioplegia.

It has been clearly shown, both in a laboratory model and in humans, that oxygenation of crystalloid cardioplegic solutions markedly enhances myocardial preservation. The addition of a small volume of red cells to a crystalloid perfusate improves capillary perfusion. Based on these results, we have changed our cardioplegic solution from cold crystalloid to cold oxygenated dilute blood. In the present study we retrospectively evaluate the results of 400 operative procedures to determine whether the addition of oxygenation and a small volume of blood to the cardioplegic solution enhances myocardial protection in the clinical setting. Two hundred consecutive patients who underwent operation with cardioplegic arrest using a cold crystalloid cardioplegic solution (group 1) were compared with a subsequent 200 patients who underwent operation with cold oxygenated dilute blood cardioplegia (group 2). Patients in group 2, who received cold oxygenated dilute blood cardioplegia, had a significantly reduced need for postoperative intraaortic balloon pump counterpulsation and for atrioventricular pacing. Also, patients in group 2 had a lower incidence of perioperative myocardial infarction and had improved early outcome. None of the 200 patients in group 2 had electrocardiographic evidence of perioperative infarction. We conclude that cold oxygenated dilute blood cardioplegia provides better preservation than does a nonoxygenated crystalloid solution during elective ischemic arrest, because a cold crystalloid solution is able to deliver oxygen and the red cells are able to enhance capillary perfusion.

Determination of clonality in acute nonlymphocytic leukemia by restriction fragment length polymorphism and methylation analysis.

Determination of cellular clonality in hematological malignancies provides fundamental information that is important in understanding the pathogenesis of these disorders. We present here an extension of one approach to accomplish this that is based on the interpretation of different methylation patterns on active and inactive X chromosomes within the region of the hypoxanthine-guanine phosphoribosyltransferase gene spanned by a restriction fragment length polymorphism. The successful application of the method to determine clonality is described for three female patients with acute nonlymphocytic leukemia.

New mutation and prenatal diagnosis in ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) is an X-linked hepatic enzyme in the urea cycle necessary for ammonia detoxification. Deficiency of OTC results in neonatal hyperammonemia, coma, and death in childhood. Because fibroblasts do not express OTC, prenatal diagnosis in the past has required fetal liver biopsy. Using a complementary DNA (cDNA) for OTC for Southern blot analysis of genomic DNA, we have found probands with complete OTC deficiency from two unrelated families in whom the same TaqI restriction endonuclease site has been altered because of independent, but not necessarily identical, mutations in the OTC gene, suggesting that this site may be a relative hotspot for mutation at a location that is critical for normal gene function. This TaqI alteration has allowed the identification of the individual in each family in whom the mutation originated as well as the exclusion of a recurrence of OTC deficiency in a male fetus at risk for the disease. OTC deficiency joins the growing list of genetic disorders for which Southern blot analysis allows accurate heterozygote detection and prenatal diagnosis in conditions for which they were not previously available.

Two anonymous X-specific human sequences detecting restriction fragment length polymorphisms in region Xq26----qter.

Two anonymous X-specific sequences isolated from a genomic library of flowsorted X chromosomal DNA were selected for study because they revealed restriction fragment length polymorphisms in the region Xq26----qter. One sequence, DXS10, detected a two-allele TaqI polymorphic system with allele frequencies of 0.33 and 0.67. The other, 4D-8, defined an MspI polymorphism with allele frequencies of 0.18 and 0.82. DXS10 is tightly linked to the hypoxanthine phosphoribosyltransferase (HPRT) locus with recombination distance theta = O cM at LOD = 5.55 (95% probability limit theta less than 15 cM). DXS10 maps to Xq26 but is not contained within the HPRT locus itself. 4D-8 shows no detectable linkage to the HPRT locus, with maximum likelihood estimate for theta = 50 cM and a LOD score of -2.61 at theta = 5 cM. These two polymorphisms provide additional chromosomal loci for gene mapping by linkage at the distal end of the long arm of the human X chromosome.

Five year experience with pancreatic pseudocysts.

Twenty-six patients with pancreatic pseudocysts underwent surgical intervention from 1975 through 1979. Chronic alcohol use was associated with pancreatic disease in 84.6 percent of these patients. The clinical findings are not specific, and ultrasonographic examination of the abdomen and computed tomographic scanning have been the most reliable diagnostic tests. External drainage is performed for infected or thin-walled cysts, and carries a complication rate of 72.7 percent in this series. Internal drainage was complicated 31 percent of the time. There were no deaths in this series.

A coronary teaching program in a community hospital.

In conclusion, the teaching program we developed appears to meet the needs originally established by the audit. Nurses are provided with a method of assessment, evaluation, and documentation. The program also provides a means of teaching continuity while following a structured time schedule that allows for recognition and satisfaction of individual patient needs. The patient teaching record provides the final tool as a reference and ongoing source of evaluation.