Whole-exome sequencing for mutation detection in pediatric disorders of insulin secretion: Maturity onset diabetes of the young and congenital hyperinsulinism.

BACKGROUND: To assess the utility of whole-exome sequencing (WES) for mutation detection in maturity-onset diabetes of the young (MODY) and congenital hyperinsulinism (CHI). MODY and CHI are the two commonest monogenic disorders of glucose-regulated insulin secretion in childhood, with 13 causative genes known for MODY and 10 causative genes identified for CHI. The large number of potential genes makes comprehensive screening using traditional methods expensive and time-consuming. METHODS: Ten subjects with MODY and five with CHI with known mutations underwent WES using two different exome capture kits (Nimblegen SeqCap EZ Human v3.0 Exome Enrichment Kit, Nextera Rapid Capture Exome Kit). Analysis was blinded to previously identified mutations, and included assessment for large deletions. The target capture of five exome capture technologies was also analyzed using sequencing data from >2800 unrelated samples. RESULTS: Four of five MODY mutations were identified using Nimblegen (including a large deletion in HNF1B). Although targeted, one mutation (in INS) had insufficient coverage for detection. Eleven of eleven mutations (six MODY, five CHI) were identified using Nextera Rapid (including the previously missed mutation). On reconciliation, all mutations concorded with previous data and no additional variants in MODY genes were detected. There were marked differences in the performance of the capture technologies. CONCLUSIONS: WES can be useful for screening for MODY/CHI mutations, detecting both point mutations and large deletions. However, capture technologies require careful selection.

Epigenetic and gene expression analysis of ankylosing spondylitis-associated loci implicate immune cells and the gut in the disease pathogenesis.

Ankylosing spondylitis (AS) is a common immune-mediated arthropathy primarily affecting the spine and pelvis. Most AS patients have subclinical intestinal inflammation, suggesting the gut microbiome and the immune response play a role in pathogenesis. Susceptibility to AS is primarily genetic, and at least 114 susceptibility variants have been identified to date. We applied bioinformatic methods utilizing epigenetic and gene and protein expression data to identify the cell types through which AS-associated variants operate. Variants were enriched in transcriptionally regulated regions in monocytes, CD4+ and CD8+ T cells, natural killer cells, regulatory T cells and B cells and mucosa from the small intestine, sigmoid colon and rectum. Weak signals were detected in bone cells, consistent with bone disease being a secondary manifestation. RNA sequencing of blood cells from AS patients and controls identified differentially expressed genes. Interrogation of expression databases showed that the upregulated genes were enriched in monocytes and downregulated genes were enriched in CD8+ T cells and natural killer cells. Gene Ontology term enrichment analysis identified microbes and the gut in the aetiology of AS. These findings identify the key immune cell types that drive the disease, and further highlight the involvement of the gut microbiome in the pathogenesis of AS.

Structural differences in chromosomes distinguish species in the tomato clade.

The tomato clade of Solanaceae is composed of 12 species that are all diploid with the same chromosome number and morphology. Species in the tomato clade are considered to have evolved primarily by genic changes rather than large-scale chromosomal rearrangements because pachytene chromosomes in F(1) hybrids synapse normally along their lengths and linkage maps of intra- and inter-specific hybrids are co-linear. However, small inversions have been reported between tomato and some of its wild relatives. Therefore, we reevaluated 5 F(1) hybrids using high-resolution, electron microscopic examination of pachytene chromosome (= synaptonemal complex) spreads to determine whether any minor structural changes had occurred among species in the tomato clade, which were not easily visible using light microscopic analysis of conventional chromosome squashes. Our study revealed a number of unexpected synaptic configurations such as mismatched kinetochores, inversion loops and reciprocal translocations. Most of these structural differences were in or close to heterochromatin that has comparatively few genes and little recombination, so they would be expected to have little effect on the evident colinearity of linkage maps, especially in euchromatin. However, these results demonstrate that substantial changes in chromosome structure have occurred among species within the tomato clade.

Role of fluorescence in situ hybridization in sequencing the tomato genome.

The tomato (Solanum lycopersicum L.) genome is being sequenced by a consortium of laboratories in 10 countries. Seventy-seven percent of the tomato genome (DNA) is located in repeat-rich, gene-poor, pericentric heterochromatin, while 23% of the genome is located in repeat-poor, gene-rich, distal euchromatin. It is estimated that approximately 90% of tomato's nuclear genes can be characterized by limiting the sequencing effort to euchromatin while avoiding the problems involved in sequencing the repetitive DNA in heterochromatin. Sequencing is being performed on tomato nuclear DNA cloned into bacterial artificial chromosome (BAC) vectors. Fluorescence in situ hybridization (FISH) is used to help direct the sequencing effort by cytologically demonstrating the location of selected BACs on tomato chromosomes. While mitotic metaphase chromosomes are too short and compact for this purpose, long pachytene chromosomes are ideal. BACs localized in euchromatin can be used confidently as anchors for the assembly of BAC contigs that extend through the euchromatic length of each chromosome arm. Another important role for FISH is identification of BACs near telomeres and near borders with pericentric heterochromatin to indicate that sequencing should not extend much further. This role of FISH is enhanced by our ability to estimate base pair distances between localized BACs and these chromosomal features. Finally, it is noteworthy that when BAC-FISH is combined with chromosomal in situ suppression (CISS) hybridization to block repeats and localize single/low copy sequences, the great majority of BACs localize to single sites. This observation is consistent with tomato being an ancient diploid.

Identification of a membrane skeleton in platelets.

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

Recombination nodules in plants.

The molecular events of recombination are thought to be catalyzed by proteins present in recombination nodules (RNs). Therefore, studying RN structure and function should give insights into the processes by which meiotic recombination is regulated in eukaryotes. Two types of RNs have been identified so far, early (ENs) and late (LNs). ENs appear at leptotene and persist into early pachytene while LNs appear in pachytene and remain into early diplotene. ENs and LNs can be distinguished not only on their time of appearance, but also by such characteristics as shape and size, relative numbers, and association with unsynapsed and/or synapsed chromosomal segments. The function(s) of ENs is not clear, but they may have a role in searching for DNA homology, synapsis, gene conversion and/or crossing over. LNs are well correlated with crossing over. Here, the patterns of ENs and LNs during prophase I in plants are reviewed.

Asymmetrical core structure in phycobilisomes of the cyanobacterium Synechocystis 6701.

The light-harvesting complex of cyanobacteria and red algae, the phycobilisome, has two structural domains, the core and the rods. Both contain biliproteins and linker peptides. The core contains the site of attachment to the thylakoid membrane and the energy transfer link between the phycobilisome and chlorophyll. There are also six rod-binding sites in the membrane-distal periphery of the core. The structure of phycobilisomes in the cyanobacterium Synechococcus 6301 was studied by Glazer, who proposed a model for the internal organization of the bicylindrical core. In the construction of that model, it was necessary to make arbitrary decisions between two possible locations for one of the trimeric protein complexes within a core cylinder and between two possible orientations of the basal core cylinders relative to one another. We isolated the tricylindrical cores from an ultraviolet-light-induced mutant of the cyanobacterium Synechocystis 6701 and obtained, by partial dissociation, a unique core substructure that maintained some contacts between the two basal cylinders. From its structure and spectral properties, we conclude that this particle is a central core substructure that resulted from dissociation of the two layers of peripheral trimers in the intact core. The compositions of this particle and the dissociated trimers were inconsistent with the proposed location of one of the trimers in the 6301 core model, but supported the placement of that trimer in the alternative position within the basal core cylinder. Rod-binding sites within the central core substructure were studied by partial dissociation of the short-rod phycobilisomes from another mutant of 6701. This dissociation generated particles that were interpreted as being central core substructures with the two basal rods attached. The appearance of these particles in the electron microscope suggested that both basal rods would be localized towards the same side of the intact core. Such an asymmetrical arrangement of basal rods is supported by previously published edge-views of intact cores with basal rods from strain 6701. These observations suggest a parallel arrangement of the basal cylinders with respect to each other, creating an asymmetrical core. A phycobilisome model was constructed that incorporated core asymmetry. This model predicts the energy transfer pathways from the basal and upper rods to specific trimers in the core.

The distribution of early recombination nodules on zygotene bivalents from plants.

Early recombination nodules (ENs) are protein complexes approximately 100 nm in diameter that are associated with forming synaptonemal complexes (SCs) during leptotene and zygotene of meiosis. Although their functions are not yet clear, ENs may have roles in synapsis and recombination. Here we report on the frequency and distribution of ENs in zygotene SC spreads from six plant species that include one lower vascular plant, two dicots, and three monocots. For each species, the number of ENs per unit length is higher for SC segments than for (asynapsed) axial elements (AEs). In addition, EN number is strongly correlated with SC segment length. There are statistically significant differences in EN frequencies on SCs between species, but these differences are not related to genome size, number of chromosomes, or phylogenetic class. There is no difference in the frequency of ENs per unit length of SC from early to late zygotene. The distribution of distances between adjacent ENs on SC segments is random for all six species, but ENs are found at synaptic forks more often than expected for a random distribution of ENs on SCs. From these observations, we conclude that in plants: (1) some ENs bind to AEs prior to synapsis, (2) most ENs bind to forming SCs at synaptic forks, and (3) ENs do not bind to already formed SCs.

Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein.

We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata.

Spontaneous platelet aggregation. Occurrence in an asymptomatic individual.

Spontaneous platelet aggregation in an asymptomatic individual is described. The platelet-poor plasma of the subject greatly enhanced the response of normal platelet-rich plasma to adenosine diphosphate. The spontaneous platelet aggregation was easily inhibited by aspirin.

Imipramine compliance in adolescents.

OBJECTIVE: To investigate side effects, medication compliance, and assumption of medication assignment in adolescents taking imipramine versus placebo in a clinical trial. METHOD: Sixty-three anxious-depressed adolescents in an 8-week double-blind study of imipramine versus placebo, each in combination with cognitive-behavioral therapy for school refusal, were evaluated. Measures of side effects, global improvement, family functioning, medication compliance based on pill counts, and guesses of drug assignment (imipramine versus placebo) were analyzed. RESULTS: Mean side effects ratings were significantly higher for the imipramine group compared with the placebo group (p = .001). Side effects were not associated with noncompliance or with dropping out. Oppositional defiant disorder (ODD) in the adolescents was significantly associated with medication noncompliance (p = .036). On the Family Adaptability and Cohesion Evaluation Scale II (FACES II), low family adaptability (i.e., rigidity), low family cohesion (i.e., disengagement), and extreme family type were significantly associated with greater noncompliance with medications. Accuracy rates for guessing medication assignment (imipramine versus placebo) were 66% for subjects, 62.5% for mothers, and 79.5% for the psychiatrist. Logistic regression demonstrated that side effects (p = .005) and global improvement scores (p = .06) predicted the psychiatrist's guesses of drug assignment. CONCLUSIONS: Side effects were not associated with noncompliance. Nonadherence with taking medications was associated with ODD in the adolescents and problematic family functioning on FACES II. The psychiatrist, who was blind to treatment condition, guessed the subjects' medication assignments with high accuracy. Thus, because of expectancy bias, the data support the use of blind independent evaluators for rating changes in medication trials.

Cardiovascular effects of gallamine triethiodide and succinylcholine chloride during halothane anesthesia in the dog.

The cardiovascular effects of gallamine triethiodide and succinylcholine chloride were studied in Beagle dogs during controlled halothane anesthesia. Small but significant increases in heart rate and mean arterial presssure were observed 1 minute after intravenous injection of succinylcholine chloride. Intravenous injection of gallamine triethiodide did not produce significant cardiovascular changes.

Effect of dobutamine in postperfusion cardiac failure in the dog.

The effect of dobutamine on cardiac function of dogs was investigated. Sixteen dogs were submitted to cardiopulmonary bypass. The aorta of each dog was cross-clamped for 1 hour; attempt was not made to perfuse the heart. After 1 hour, 8 of the 16 dogs were randomly selected and treated with dobutamine (5 mug/kg/min). The other 8 dogs were designated the control group and were given placebo. Postperfusion failure and death were used as end point criteria. Dogs given dobutamine responded remarkably well, with significantly decreased postperfusion low output syndrome. Evidence of cardiac function 5 minutes after the removal of the bypass was the criterion used to determine survival of the surgical cross-clamp procedure; however, this did not necessarily indicate survival of the dog. Of the 8 dogs given dobutamine, 6 (75%) survived the surgical cross-clamp, whereas of the 8 dogs not given dobutamine, 3 (37.5%) survived the surgical procedure. Seemingly, the effect of dobutamine is not mainly chronotropic, but is rather a direct aid to myocardial strength.

Aortic regurgitation in the dog.

In a review of aortic regurgitation in 12 dogs, breed or sex predilection was not found. Clinical signs included decreased exercise tolerance and diastolic murmur. Associated anomalies included ventricular septal defect and aortic stenosis. It was concluded that when aortic regurgitation develops secondary to a ventricular septal defect, the prognosis should be grave.

Myocardial function in small dogs with chronic mitral regurgitation and severe congestive heart failure.

Systolic myocardial function was assessed in 16 dogs with severe congestive heart failure due to chronic mitral valve fibrosis. End-systolic diameters were measured on echocardiograms and end-systolic volume indices were calculated. Thirteen of the 16 dogs (81%) had normal or only mildly abnormal myocardial function. These data suggested that myocardial failure is not a prominent factor contributing to signs of heart failure in dogs with mitral regurgitation. Because of these data, the routine use of digitalis glycosides to increase cardiac contractility is seriously questioned in dogs with heart failure secondary to chronic mitral regurgitation.

Efficacy of digoxin administration in dogs with idiopathic congestive cardiomyopathy.

Digoxin administration (0.22 mg/m2 of body surface BID) to 10 large-breed dogs with congestive cardiomyopathy increased shortening fraction more than 5.5% in 4 of the dogs. This group of dogs lived longer than the group that did not have a positive inotropic response to digoxin. Heart rate decreased in both groups of dogs. Base-line jugular PVO2 were low in all dogs. Jugular PVO2 decreased significantly in the group that did not respond to digoxin, presumably because of decreased cardiac output. Jugular PVO2 consistently increased in dogs that had a positive inotropic response to digoxin. Base-line shortening fraction, heart rate, and PVO2 did not predict which dogs would respond to digoxin. Serum digoxin concentrations were consistently between 1.5 and 2.5 ng/ml. It was concluded that digoxin administration is not efficacious in all dogs with congestive cardiomyopathy and that the positive inotropic response is not predicted by base-line shortening fraction, heart rate, or jugular PVO2. Dogs that do respond to digoxin usually live longer than those that do not. Jugular PVO2 can be used to separate dogs that do respond from dogs that do not respond to digoxin as long as the base-line PVO2 is low. The negative chronotropic effects of digoxin may be detrimental to dogs that do not have a positive inotropic effect from digoxin.

Pulmonary artery banding for ventricular septal defect in dogs and cats.

Five dogs and 1 cat had pulmonary artery banding for ventricular septal defect and congestive heart failure. An umbilical tape band constricted the pulmonary artery to one-third its original diameter, increased the right ventricular pressure, and decreased the left ot right shunt. Five animals had remission of clinical signs; 1 dog died of right ventricular failure due to a band that had been applied too tightly. The results indicated pulmonary artery banding is helpful in reduction of clinical signs, due to increased flow to the lungs from ventricular septal defect.

Surgical repair of atrial septal defect in a dog.

Atrial septal defect, a relatively rare canine congenital cardiovascular defect, caused syncope during excitement in a 5-year-old male Boxer dog. The defect was successfully repaired by open heart surgery.

Total quality management approach improves medication replacement.

Total quality management (TQM) is based on understanding customer needs, improving key processes that affect customer satisfaction, and creating cross-functional teams to resolve process problems. This article describes application of TQM principles and problem-solving processes to improve one OR's medication exchange system. The problem was excessive monthly pharmacy medication replacement costs. The goal was to reduce the monthly medication replacement costs by 50%. Within four months, monthly medication replacement charges decreased from $656 to $302, and by one year, monthly charges decreased to $160. The new process had fewer steps, fewer staff members involved, and fewer delays in medication replacement.

Orientation based on nursing diagnoses. Old concepts in today's practice.

Although many operating room orientation programs contain content necessary to meet accrediting guidelines, very few tie the nursing process to the content. Our orientation is structured within a nursing framework (ie, Dr Gordon's "Eleven Functional Health Patterns") and emphasizes nursing diagnoses, theory, and clinical competencies. Although the new orientation program has been in effect for only two years, we feel the following list reflects the positive outcomes so far: decreased staff turnover (ie, one nurse out of 26 full-time equivalents in 18 months), increased success in recruiting nurses into the operating room (ie, multiple applicants as positions open), new nurses demonstrate comfort with basic perioperative nursing practice with-in six months, and nurses who did not complete new orientation program are requesting all or portions of the content. By using this plan, essential aspects of perioperative practice are consistent with hospital-wide nursing practice, practice standards for the operating room, and accrediting standards.